Rapid detection of microorganisms

ABSTRACT

Tools and methods for detecting the presence bacteria, yeast and mold in a sample obtained from a food sample are provided. The methods employ a polymerase chain reaction and primer and probe sets that are based on the 16S rRNA and squalene-hopene cyclase genes of  Alicyclobacillus  and  Geobacillus  and the 18S rDNA gene of mold and yeast. The present invention also relates to primer and probe sets. Each primer and probe set comprises a forward primer and a reverse primer, both of which are from 15 to 35 nucleotides in length and a probe.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Applications No. ______, filed Oct. 22, 2003, No. 60/500,736, filed Sep. 5, 2003, and No. 60/430,202, filed Dec. 2, 2002, each of which is incorporated herein by reference in their entirety.

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention provides methods and tools for rapidly detecting microorganisms such as molds and fungi, and acid and thermophilic Alicyclobacillus spp and Geobaillus spp. in test samples, particularly food samples.

BACKGROUND

[0003] Spoilage of products, particularly food and beverage products, due to contamination with bacteria, yeasts and molds, results in significant financial loss to the food industry. Yeasts and molds are commonly associated with raw materials of foods and are often found in the processing environment. Due to the structural features of both the vegetative cells and spores of fungi, these food contaminants have a good chance of surviving current processing conditions. Yeasts and molds can grow within a wide range of environmental conditions, and therefore the presence in food of even minor amounts of yeast and mold contaminants can cause spoilage during storage.

[0004] Like fungi, many bacteria are resitant to processing conditions, and some are resistant even to high acid conditions in food and beverage products. Alicyclobacilli are Gram-positive, spore-forming, aerobic rods classified as thermoacidophiles capable of growing at high temperatures and low pH (1, 2, 3). These bacteria, formerly of the Bacillus genus, were assigned into the new genus Alicyclobacillus in 1992 (1). Sequence analysis of the 16s rRNA genes proved that three previously classified Bacillus thermoacidophiles (B. acidocaldarius, B. acidoterrestris, and B. cycloheptanicus) belong in a group that differs from other closely related Bacilli. Additionally, a key phenotypic variation was found in the membrane composition of these three species. The primary fatty acid component in the membrane was determined to be ω-alicyclic fatty acids, a type of lipid not found in other Bacillus species at the time. This evidence initiated the establishment of the Alicyclobacillus genus of obligate acidothermophiles, containing A. acidocaldarius, A. acidoterrestris, and A. cycloheptanicus, within the Bacillus branch (1). More recently, A. hesperidum and Alicyclobacillus genomic species 1 and 2 (24, 25), A. acidiphilus (22), A. herbarius (23), A. sendaiensis (26), and A. pomorum (27) have been added as new species within the genus Alicyclobacillus.

[0005] Alicyclobacilli have been an increasingly frequent spoilage problem in the beverage industry, particularly acidic juices, during the last two decades. In 1982, a Bacillus sporeformer (to be later classified as B. acidoterrestris and then subsequently A. acidoterrestris) capable of growing at pH as low as 2.5 was isolated from apple juice (4, 5, 6). In 1994, Splittstoesser et al. discovered the presence of A. acidoterrestris in apple juice, further shown by Yamazaki et al. in 1996 (7, 8). Spore germination and growth in orange juice (3) and grapefruit juice (6) was even observed. White grape juice, tomato juice, cranapple juice, and pear juice have also been afflicted with Alicyclobacillus spoilage (11).

[0006] While Alicyclobacilli are non-pathogenic, they are a spoilage agent that can drastically affect the quality of acidic fruit juices. Pettipher et al. (1997) reported that guiacol, one of the chemicals responsible for the off-odor and smoky taints characteristic in Alicyclobacillus-spoiled juices, can be detected by taste before any visible contamination is seen (3). Therefore, a consumer would generally not be able to identify Alicyclobacillus-spoiled juice until it is ingested. In addition to guiacol, 2,6-dibromophenol (2,6-DBP) and 2,6-dichlorophenol (2,6-DCP) were found to contribute to disinfectant taints at detectable levels after as little as one day at 44° C. in containers with large headspaces. More realistically, commercially stored shelf stable juices with generally low headspace volume develop these taints within the first month of storage, particularly in warmer climates (10). The presence of these chemicals in Alicyclobacillus-spoiled juices significantly reduces the quality of the product, subsequently lowering the consumer image of the brand.

[0007] Alicyclobacilli are very heat resistant, growing from pH 2.5-5.5 and 25° C.-60° C. (6). Beyond growth, cells and spores can survive normal pasteurization procedures, at temperatures up to 97° C. (3,6). Fruit juices that are fresh squeezed, pasteurized, or hot-filled are most easily affected by Alicyclobacillus spoilage, since ultra high temperature treatment is normally sufficient for killing all microorganisms (3). Since Alicyclobacilli can survive temperatures that exceed industry standard pasteurization specifications, contamination occurring before or during the processing steps can lead to spoilage in the final product that reaches the consumer. Since significant increases in pasteurization temperatures or times ultimately affect product quality and flavor, companies aren't likely to change current procedures.

[0008] Early detection, i.e., before products reach the consumer, of the presence of even small amounts of these microbial contaminants in food and beverages is highly desirable in the food industry. Classic culture methods are generally accurate for detecting the presence of microorganisms, but can take up to a week for the results. Previdi et al. (1997) reported a method for detecting A. acidocaldarius in juice products. This method required juices or concentrates to be heat treated and then incubated at 37° C. for 7 days, followed by plating on pH 4.0 malt extract agar (13). Pinhatti et al. (1997) tested frozen orange juice concentrate by heat shocking the samples at 80° C., enriching at 50° C. for 24 and 48 h, and finally pour plating in BAM and incubating at 50° C. for 24 h (12). Both of these methods of detection provided accurate results, but took from 3-7 days to complete. As with bacteria, it can often take one to two weeks just to grow yeast and mold cells on culture media. In addition, there are so many varieties of molds and yeasts with diverse growth requirements that it is very difficult to find an optimal medium to capture all potential yeast and mold contaminants at the same time. For food industry applications, it is desirable to have a rapid detection system that does not require time consuming culture techniques to detect the presence of microbial contamination of food samples. Accordingly, it is desirable to have a more rapid detection method that can provide results within a few hours, with the same level reliability of culture methods. It is also desirable to have kits that can differentiate between specific types of microbes and which comprise microbe-specific reagents that are useful for conducting rapid sample testing.

SUMMARY OF THE INVENTION

[0009] The present invention provides methods and kits for detecting the presence of Alicyclobacillus spp. and a closely related thermophilic bacterium, Geobacillus, in samples, particularly food samples. In one embodiment the method comprises, collecting bacterial cells in the sample, extracting DNA from the cells, and assaying for the presence of these bacterium species using a PCR technique, preferably real-time PCR, and three signature oligonucleotides (2 primers and a probe) directed to a particular sequence in a target gene encoding either the 16S rRNA or squalene-hopene cyclase (shc). (See the conserved sequences extending from nucleotide position 334 through nucleotide position 485, and from nucleotide position 752 through nucleotide position 813 of the shc gene sequence of Alicyclobacillus shown in FIG. 5. Also see the conserved sequences extending from nucleotide position 1327 through nucleotide position 1460 of the 16S rRNA gene sequence of Alicyclobacillus shown in FIG. 1.) The presence of multiple Alicyclobacillus spp. and a closely related thermophilic bacterium Geobacillus can be achieved within 3-5 hours using the described sample preparation procedures, and proper combination of the three oligonucleotides as primer-and-probe set in the real-time PCR reaction.

[0010] The kits of the present invention comprise at least one forward primer and one reverse primer, with or without a probe for amplifying a sequence of at least 50 consecutive nucleotides within a conserved region of the three Alicyclobacillus spp. shown in FIG. 1 (sequences shown in alignment). FIGS. 2, 3 and 4, respectively, show the full coding sequences for the 16S rRNA genes fromo the Alicyclobacillus strains deposited with the ATCC as 43030, 49025, and 49029. In certain embodiments, the oligonucleotides comprise the entire or a majority of the following sequences or their reverse complement sequences, as a set or as combination crossing multiple sets, e.g. in certain cases the forward primer of one set can be combined with a reverse primer that is based on the forward primer of another set. Thus the following embodiments can be used in various primer, probe, or primer-probe combinations. Depending on the primers that are combined, the lower oligo may be used as a probe. The sequence of the lower oligo corresponds to the coding sequence of the target region of the gene, and is complementary to the reverse primer in each set. The reverse primers are shown as the reverse complement of the targeted region of the gene. The forward primers correspond to the coding sequence of the target region of the gene. TABLE I Signature Oligonucleotides Directed Toward 16S rRNA gene Length Tm(° C.) GC % Set 1: Forward primer: 5′GAGCCCGCGGCGCATTAGC3′ 19 68.9 73.7 (SEQ ID NO 1) Probe: 5′GCGACGATGCGTAGCC(G)3′ 16 61.8 68.8 (SEQ ID NO 2) Lower Oligo: 5′CGCAATGGGCGCAAGC3′ 16 61.8 68.8 (SEQ ID NO 3) Reverse primer: 5′GCTTGCGCCCATTGCG3′ 16 61.8 61.8 (SEQ ID NO 4) Set 2: Forward primer: 5′GAGCAACGCCGCGTGAGCG3′ 19 68.8 73.7 (SEQ ID NO 5) Probe: 5′CTTCGGGTTGTAAAGC3′ 16 54.2 50 (SEQ ID NO 6) Lower Oligo: 5′CGGCTAACTACGTGC3′ 15 56.2 60 (SEQ ID NO 7) Reverse primer: 5′GCACGTAGTTAGCCG5′ 15 56.2 60 (SEQ ID NO 8) Set 3: Forward Primer: 5′AGTGCTGGAGAGGCAAGG3′ 18 62.2 61.1 (SEQ ID NO 9) Probe: 5′CTGGACAGTGACTGACG3′ 17 59.6 58.8 (SEQ ID NO 10) Lower Oligo 5′GCACGAAAGCGTGGGGAGCA 20 66.6 65 (SEQ ID NO 11) Reverse Primer: 5′TGCTCCCCACGCTTTCGTGC5′ 20 66.6 65 (SEQ ID NO 12) Set 4: Forward Primer: 5′GGAGTACGGTCGCAAGACTG3′ 20 64.5 60 (SEQ ID NO 13) Probe: 5′CGCACAAGCAGTGGAGC3′ 17 62.0 64.7 (SEQ ID NO 14) Lower Oligo: 5′CAGGGCTTGACATC3′ 14 52.6 57.1 (SEQ ID NO 15) Reverse Primer: 5′GATGTCAAGCCCTG3′ 14 52.6 57.1 (SEQ ID NO 16) Set 5: Forward primer: 5′GGCGTAAGTCGGAGGAAGG3′ 19 64.5 63.2 (SEQ ID NO 17) Probe: 5′ATGTCCTGGGCTACACACG3′ 19 62.3 57.9 (SEQ ID NO 18) Reverse primer: 5′GCCTGCAATCCGAACTACC5′ 19 62.3 57.9 (SEQ ID NO 19) Set CC16S: Forward primer: 5′CGTAGTTCGGATTGCAGGC3′ 19 65.6 57.9 (SEQ ID NO 20) Probe: 5′CGGAATTGCTAGTAATCGCG3′ 20 57.9 47.4 (SEQ ID NO 21) Lower Oligo: 5′CACGAGAGTCGGCAACAC3′ 18 63.3 61.1 (SEQ ID NO 22) Reverse primer: 5′GTGTTGCCGACTCTCGTG3′ 18 62.2 61.1 (SEQ ID NO 23) Set 6: primer: 5′GATGATTGGGGTGAAG3′ 16 54.2 50 (SEQ ID NO 24)

[0011] TABLE II Signature Oligonucleotides Directed Toward squalene-hopene cyclase (shc) gene These three oligonucleotides were further used as PCR primer pair and DNA probe in real-time PCR detection of Alicyclobacillus spp. Forward Primer: 5′ ATGCAGAGYTCGAACG 3′ (SEQ ID NO 25) Probe: 5′ 6-FAM d [TCG(A)GAA(G)GACGTCACCGC] BHQ-1 3′ (SEQ ID NO 26) Reverse Primer: 5′ AAGCTGCCGAARCACTC 3′ (Y = C + T; R = A + G (SEQ ID NO 27)

[0012] TABLE III The Sequence, GC % and Tm of Primer and probe set candidate 1 for Shc Gene: Name Sequence Length Tm GC % Forward primer TACTGGTGGGGGCCGCT (SEQ ID NO 28) 17 64.84 70.59 TACTGGTGGGCGCCGCT (SEQ ID NO 29) 17 64.84 70.59 Probe ATGGAAGCGGAGTACGTCC (SEQ ID NO 30) 19 62.64 57.9 ATGGAAGCGGAGTACGTCCT (SEQ ID NO 31) 20 62.45 55 ATGGAAGCGGAATATGTGC (SEQ ID NO 32) 19 58.32 47.37 ATGGAAGCGGAATATGTGCT (SEQ ID NO 33) 20 58.35 45 Reverse Primer CGCGAGGACGGCAC (SEQ ID NO 34) 14 62.11 78.57 CGCGAGGACGGCACGTGG (SEQ ID NO 35) 18 69.79 77.78 CGCGAAGACGGCAC (SEQ ID NO 36) 14 59.16 71.43 CGCGAAGACGGCACCTGG (SEQ ID NO 37) 18 67.51 72.22

[0013] TABLE IV The Sequence, GC % and Tm of Primer and probe set candidate 2 for Shc Gene: Name Sequence Length Tm GC % Forward primer CAAAAGGCGCTCGACTG (SEQ ID NO 38) 17 60.02 58.82 CAAAAGGCGCTCGACTGG (SEQ ID NO 39) 18 62.96 61.11 CAAAAGGCGCTCGACTGGGTCG (SEQ ID NO 40) 22 68.99 63.64 CAAAAGTCGCTCGACTG (SEQ ID NO 41) 17 57.61 52.94 CAAAAGTCGCTCGACTGG (SEQ ID NO 42) 18 60.68 55.56 CAAAAGTCGCTCGACTGGCTCG (SEQ ID NO 43) 22 67.13 59.09 Probe GGACGGCGGCTGGGGCGA (SEQ ID NO 44) 18 72.07 83.33 GGACGGCGGCTGGGGCGAGGA (SEQ ID NO 45) 21 75.09 80.95 GGACGGCGGCTGGGGCGAGGACTGCCG (SEQ ID NO 46) 27 80.31 81.48 GGATGGCGGTTGGGGTGA (SEQ ID NO 47) 18 65.23 66.67 GGATGGCGGTTGGGGTGAAGA (SEQ ID NO 48) 21 67.28 61.91 GGATGGCGGTTGGGGTGAAGATTGCCG (SEQ ID NO 49) 27 72.72 62.96 Reverse Primer^(a) TGATGGCGCTCATCGC (SEQ ID NO 50) 16 59.53 62.5 1 TGATGGCGCTCATCGCGGGCGGC (SEQ ID NO 51) 23 74.2 73.91 2 ACCCCGTCGCAGACGGCCTGGGCGC (SEQ ID NO 52) 25 77.7 80 3 ACACCGTCGCAGACCGCCTGGGCGT (SEQ ID NO 53) 25 74.42 72

[0014] The present invention also provides methods and kits for detecting the presence of yeast and mold contaminants in samples, particularly in food samples. In one aspect, the method comprises collecting particulate matter, preferably cells and cellular fragments, in the sample, extracting DNA from the particulate matter, and assaying for the presence of yeast DNA in the extracted DNA using a PCR technique using primers that amplify a select conserved region in 18s rDNA of representative yeast species, including Zygosaccharomyces bailii (Lindner) Guilliermond strain ATCC 36947 and the other yeast species shown FIG. 7. (See conserved sequence extending from nucleotide 81 through nucleotide 225 of the sequence of Z. bali.) Preferably, the method uses real-time PCR, and three signature oligonucleotides (2 primers and a probe) directed to a particular sequence in the gene encoding the yeast 18S rDNA.

[0015] In another aspect, the kit of the present invention comprise at least one forward primer and one reverse primer, with or without a probe for amplifying a sequence of at least 50 consecutive nucleotides within the select region of yeast 18s rDNA. In one embodiment, the kit comprises primers and a probe having the following sequences: Yupreal: 5′ GTGGTGCTAGCATTTGCTG 3′ (SEQ ID NO 54) Ylowreal: 5′ GTTAGACTCGCTGGCTCC 3′ (SEQ ID NO 55) Yprobe: 5′ TTTCAAGCCGATGGAAGTTTGA(C/G)3′ (SEQ ID NO 56)

[0016] Another probe that may be used in the present method has the following sequence 5′ CGGTTTCAAGCCGATGGAAGT 3′. (SEQ ID NO 57)

[0017] Yet another set of primers and probe for yeast detection: Oligo name Len Pur Scale Sequence (5′-3′) 18srRNA-newup-112503-1 30 DST 0.05 CCTACTAAATAGGGTGCTAGCATTTGCTGG (SEQ ID NO 58) 18srRNA-newup-112503-2 26 DST 0.05 CTAAATAGGGTGCTAGCATTTGCTGG (SEQ ID NO 59) 18srRNA-probe2 25 CGGTTTCAAGCCGATGGAAGTTTGA (SEQ ID NO 60)

[0018] In another aspect the present method comprises collecting particulate matter, preferably cells and cellular fragments, in the sample, extracting DNA from the particulate matter, and assaying for the presence of mold DNA in the extracted DNA using a PCR technique using primers that amplify a select conserved region in 18s rDNA of the following representative molds: Byssochlamys fulva Olliver et Smith, teleomorph ATCC 24474 and Penicillium digitatum Saccardo, anamorph ATCC10030, as shown in the attached alignment. (See the conserved sequence extending from nucleotide 114 through nucleotide 239 of the 18s rDNA sequence of P. digitatum shown in FIG. 7.) Preferably, the method uses real-time PCR, and three signature oligonucleotides (2 primers and a probe) directed to a particular sequence in the gene encoding the mold 18s rDNA.

[0019] In another aspect, the present the kits of the present invention comprise at least one forward primer and one reverse primer, with or without a probe for amplifying a sequence of at least 50 consecutive nucleotides within the select region of mold 18s rDNA. In one embodiment, the kit comprises primers and a probe having the following sequences: Mupreal: 5′ CCGCTGGCTTCTTAGGG 3′ (SEQ ID NO 61) Mlowreal: 5′ AGGGCCAGCGAGTACATCA 3′ (SEQ ID NO 62) Mprobe: 5′ CTCAAGCCGATGGAAGTGCG 3′ (SEQ ID NO 63)

[0020] The invention further provides a method for detecting through real-time PCR using at least one of the nucleic acid primer pairs, and at least one probe, the presence of acidophilic bacterium in a test sample, especially in a food sample. In one embodiment, the acidophilic bacterium detection method includes use of one forward primer directed to the 16S rRNA gene, wherein the primer is selected from the forward primers listed in Table I, one reverse primer directed to the 16S rRNA gene wherein the primer is selected from the reverse primers listed in Table I, and one probe directed to a sequence that is located between the sequences to which the forward and reverse primers are directed, wherein the probe is selected from the group of probles listed in Table I.

[0021] In another embodiment, the acidophilic bacterium detection method includes use of one forward primer directed to the squalene-hopene cyclase gene, wherein the forward primer is selected from the group of forward primers listed in Tables II and III, one reverse primer directed to the squalene-hopene cyclase gene wherein the primer is selected from the group of reverse primers listed in Tables II and III, and one probe directed to a sequence that is located between the sequences to which the forward and reverse primers are directed, wherein the probe is selected from the group of probes listed in Tables II and III.

[0022] In yet another embodiment, the acidophilic bacterium detection method includes use of one forward primer directed to the squalene-hopene cyclase gene, wherein the forward primer is selected from the group of forward primers listed in Table IV, one reverse primer directed to the squalene-hopene cyclase gene wherein the primer is selected from the group of reverse primers listed in Table IV, and one probe directed to a sequence that is located between the sequences to which the forward and reverse primers are directed, wherein the probe is selected from the group of probes listed in Table IV.

[0023] In another embodiment, the yeast detection method includes use of one forward primer directed to the 18S rDNA gene, wherein the primer is selected from the group consisting of SEQ ID NO 54 and SEQ ID NO 58, one reverse primer directed to the 18S rDNA gene wherein the primer is selected from the group of consisting of SEQ ID NO 55 and SEQ ID NO 55, and one probe directed to a sequence that is located between the sequences to which the forward and reverse primers are directed, wherein the probe is selected from the group consisting of SEQ ID NO 56, SEQ ID NO 57 and SEQ ID NO 60.

[0024] In yet another embodiment, the mold detection method includes use of one forward primer directed to the 18S rDNA gene, wherein the primer corresponds to SEQ ID NO 61, one reverse primer directed to the 18S rDNA gene wherein the primer corresponds to SEQ ID NO 62, and one probe directed to a sequence that is located between the sequences to which the forward and reverse primers are directed, wherein the probe corresponds to SEQ ID NO 63.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 shows polynucleotide sequence alignmnent of 16S rRNA gene fragments from three representative strains of Alicyclobacillus, specifically, A. acidocaldarius ATCC43030, A. acidoterrestris ATCC49025, and A. cycloheptanicus ATCC49029

[0026]FIG. 2 shows the 16S rRNA gene coding Sequence for A. cycloheptanicus ATCC49029

[0027]FIG. 3 shows the 16S rRNA gene coding Sequence for A. acidoterrestris ATCC49025

[0028]FIG. 4 shows the 16S rRNA gene coding Sequence for A. acidocaldarius ATCC43030

[0029]FIG. 5: shows the Shc gene sequence alignments for A. cycloheptanicus ATCC49029 and A. acidoterrestris ATCC49025

[0030]FIG. 6: shows the Shc amino acid sequence alignments for A. cycloheptanicus ATCC49029 and A. acidoterrestris ATCC49025

[0031]FIG. 7 shows the alignment for the 18s rDNA gene coding Sequence for Zygosaccaromyces, Penecillium digitatum, and Byssochlamys fulva

[0032]FIG. 8 shows the 16S rRNA gene coding sequence alignments for several strains of for A. cycloheptanicus

[0033]FIG. 9. shows the results of Real-time PCR detection of A. acidocaldarius (black), A. cycloheptanicus (blue), and A. acidoterrestris (lt. green) using the CC16S specific probe and primer pair.

[0034]FIG. 10 shows the results of Real-time PCR sensitivity test of A. acidoterrestris using the CC16S primers and probe

[0035]FIG. 11 shows the results of Real-time PCR sensitivity test of A. acidoterrestris in orange juice.

[0036]FIG. 12 shows the 18s rDNA gene coding Sequence for Zygosaccaromyces

[0037]FIG. 13 shows the 18s rDNA gene coding Sequence for Penecillium digitatum

[0038]FIG. 14 shows the 18s rDNA gene coding Sequence for Byssochlamys fulva

[0039]FIG. 15 shows the results of a specificity test. • Zygosaccharomyces bailii (Lindner) Guilliermond ATCC 36947; ▪ industry sample yeast. ♦ Byssochlamys fulva Olliver et Smith ATCC 24474; ▾ H₂O control with extraction. ▴ H₂O control without extration.

[0040]FIG. 16 shows the results of a specificity test with ▾ yeast, ♦ mold and acciobacillus and ▴ H₂O

[0041]FIG. 17 shows the results of a specificity test with ▪ Z.b (yeast).; ▴ B.F. (mold); ♦ Accidobacillus; ▾ water; Apple; ▾ green grape; and ▪ Red grape.

[0042]FIG. 18 shows the results of a specificity test with ▪ Z.b.; ▴ B.F.; ♦ Accidobacillus and ▾ water.

[0043]FIG. 19 shows the results of a specificity test with ▪ Orange1; ▴ Orange2; ♦ Orange Juice Supernatant; • Orange Juice pellet; • Yeast; and ▾ H₂O

[0044]FIG. 20 shows the results of a specificity test with !Byssochlamys fulva Olliver et Smith, telomorph ATCC 24474; Penicillium digitatum Saccardo, anamorph ATCC 10030; #Zygosaccharomyces bailii (Lindner) Guillermond, telomorph deposited as Saccharomyces bailii Lindner, telomorph ATCC 36947; % Industry Mold 42; &Indusrty Mold 41; “Industry Mold 3; “Water (extracted); % water (not extracted)

[0045]FIG. 21 shows specificity test results with Bussochlamys fulva Olliver et Smith, teleomorph ATCC24474; water and Zygosaccharomyces bailii (Lindner) Guilliermond, telomorph depositied as Saccharomyces bailii Lindner, telomorph ATCC 36947; Acidobacillus acidoterrestris 49025.

[0046]FIG. 22 shows the Alignment^(a) of 134 bp priming region flanked by CC16S-F (CGTAGTTCGGATTGCAGGC), CC16S-Probe (CGGAATTGCTAGTAATCGC), and CC16S-R (CACGAGAGTCGGCAACAC)^(b).

[0047]FIG. 23 shows the results of Real-time PCR detection of A. acidocaldarius ATCC 43030 (•), A. cycloheptanicus ATCC 49029 (♦), and A. acidoterrestris ATCC 49025 (▪) using the CC 16S primer and probe set.

[0048]FIG. 24 shows the results of Real-time PCR sensitivity test of A. acidoterrestris ATCC 49025 in saline solution using the CC16S primers and probe

[0049]FIG. 25 shows the results of Real-time PCR sensitivity test of A. acidoterrestris ATCC 49025 in orange juice, using the CC16S primers and probe.

[0050]FIG. 26 shows the results of Real-time PCR detection of food-borne microorganisms using the developed primer-and-probe set.

[0051]FIG. 27 shows the resuls f Real-time PCR sensitivity test

[0052]FIG. 28. Real-time PCR detection of A. acidocaldarius ATCC43030 cells in apple juice using shr-specific primer-and-probe set.

DETAILED DESCRIPTION OF THE INVENTION

[0053] The methods and kits provided herein enable the rapid and reliable detection of contaminating microorganisms that are found in test samples of products, preferably consumer products, and most preferably food products. The methods are especially suited for the detection of Alicyclobacillus spp. including A. acidocaldarius, A. acidoterrestris, A. cycloheptanicus, A. hesperidum, A. acidiphilus, A. herbarius, A. sendaiensis, and A. pomorum and Geobacillus stearothemophilus, and a variety of yeasts and mold. Other reported methods use conventional PCR (using a pair of oligonucleotides as primers) to detect the presence of Alicyclobacillus spp. (Obara and Niwa, 1998) which usually is associated with the problem of high background with non-specific PCR products.

[0054] According to the methods described herein, a sample is obtained from a test material, for example a sample of a fruit juice or other food product. The sample is processed to extract any polynucleotides in the sample, particularly polynucleotides from target organisms that may be present in the material. After extraction and processing according to methods described herein or otherwise known in the art, the sample is treated with reagents that comprise a forward primer oligonucleotide, a reverse primer oligonucleotide, and a labeled oligonucleotide probe, wherein the reagents are targetted for specific regions within the genome of target organisms. The sample is then processed according to PCR amplification methods. The PCR product is first amplified using the primers. Binding of the labeled probe to a target sequence within the PCR product that corresponds with a target region in the genomic DNA of the contaminating bacteria or mold signals the presence of contaminating microorganisms.

[0055] Therefore the combination of the three unique sequences and the real-time PCR technology ensured specific and sensitive detection of the presence of the target bacteria. This real-time PCR approach also offers other features such as a) accuracy: more than one probe will be included in the detection system with less possible error; b) flexibility: up to four PCR products can be simultaneously detected so potentially incorporating probes for other spoilage microorganisms into the detection system is expected.

[0056] Primer Selection

[0057] Primers are selected within the conserved regions shown in the attached alignment (FIG. 1) to amplify a fragment with proper size for optimal detection. One primer is located at each end of the sequence to be amplified. Such primers will normally be between 10 to 35 nucleotides in length and have a preferred length from between 18 to 22 nucleotides. The smallest sequence that can be amplified is approximately 50 nucleotides in length (e.g., a forward and reverse primer, both of 20 nucleotides in length, whose location in the sequences is separated by at least 10 nucleotides). Much longer sequences can be amplified. Preferably, the length of sequence amplified is between 75 and 250 nucleotides in length, and between 75 and 150 for Taqman assay.

[0058] One primer is called the “forward primer” and is located at the left end of the region to be amplified. The forward primer is identical in sequence to a region in the top strand of the DNA (when a double-stranded DNA is pictured using the convention where the top strand is shown with polarity in the 5′ to 3′ direction). The sequence of the forward primer is such that it hybridizes to the strand of the DNA which is complementary to the top strand of DNA.

[0059] The other primer is called the “reverse primer” and is located at the right end of the region to be amplified. The sequence of the reverse primer is such that it is complementary in sequence to, i.e., it is the reverse complement of a sequence in, a region in the top strand of the DNA. The reverse primer hybridizes to the top strand of the DNA.

[0060] PCR primers should also be chosen subject to a number of other conditions. PCR primers should be long enough (preferably 10 to 30 nucleotides in length) to minimize hybridization to greater than one region in the template. Primers with long runs of a single base should be avoided, if possible. Primers should preferably have a percent G+C content of between 40 and 60%. If possible, the percent G+C content of the 3′ end of the primer should be higher than the percent G+C content of the 5′ end of the primer. Primers should not contain sequences that can hybridize to another sequence within the primer (i.e., palindromes). Two primers used in the same PCR reaction should not be able to hybridize to one another. Although PCR primers are preferably chosen subject to the recommendations above, it is not necessary that the primers conform to these conditions. Other primers may work, but have a lower chance of yielding good results.

[0061] PCR primers that can be used to amplify DNA within a given sequence can be chosen using one of a number of computer programs that are available. Such programs choose primers that are optimum for amplification of a given sequence (i.e., such programs choose primers subject to the conditions stated above, plus other conditions that may maximize the functionality of PCR primers). One computer program is the Genetics Computer Group (GCG recently became Accelrys) analysis package which has a routine for selection of PCR primers. There are also several web sites that can be used to select optimal PCR primers to amplify an input sequence. One such web site is http://alces.med.umn.edu/rawprimer.html. Another such web site is http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi.

[0062] Making the Oligonucleotide Primers and Probes

[0063] The oligonucleotide primers and probes disclosed in this application can be made in a number of ways. One way to make these oligonucleotides is to synthesize them using a commercially-available nucleic acid synthesizer. A variety of such synthesizers exists and is well known to those skilled in the art. Many such synthesizers use phosphoramidite chemistry, although other chemistries can be used. Phosphoramidite chemistry utilizes DNA phosphoramidite nucleosides, commonly called monomers, to synthesize the DNA chain or oligonucleotide. Such monomers are modified with a dimethoxytrityl (DMT) protecting group on the 5′-end, a b-cyanoethyl protected 3′-phosphite group, and may also include additional modifiers that serve to protect reactive primary amines in the heterocyclic ring structure (to prevent branching or other undesirable side reactions from occurring during synthesis).

[0064] To make an oligonucleotide of a specific sequence, phosphoramidite nucleosides are added one-by-one in the 3′-5′ direction of the oligonucleotide, starting with a column containing the 3′ nucleoside temporarily immobilized on a solid support. Synthesis initiates with cleavage of the 5′-trityl group of the immobilized 3′ nucleoside by brief treatment with acid [dichloroacetic acid (DCA) or trichloroacetic acid (TCA) in dichloromethane (DCM)] to yield a reactive 5′-hydroxyl group. The next monomer, activated by tetrazole, is coupled to the available 5′-hydroxyl and the resulting phosphite linkage is oxidized to phosphate by treatment with iodine (in a THF/pyridine/H₂O solution). The above describes the addition of one base to the oligonucleotide. Additional cycles are performed for each base that is added. The final oligonucleotide added does not have a 5′ phosphate. When synthesis is complete, the oligonucleotide is released from the support by ammonium hydroxide and deprotected (removal of blocking groups on nucleotides).

[0065] Normally, oligonucleotides of up to 150-180 bases long can be efficiently synthesized by this method using a nucleic acid synthesizer. To make oligonucleotide that are longer than 100 bases, two single-stranded oligonucleotides, that are partially complementary along their length, can be synthesized, annealed to one another to form a duplex, and then ligated into a plasmid vector. Once a plasmid containing the ligated duplexes has been formed, additional oligonucleotide duplexes can be ligated into the plasmid, adjacent to the previously ligated duplexes, to form longer sequences. It is also possible to sequentially ligate oligonucleotide duplexes to each other, to form a long, specific sequence, and then clone the single long sequence into a plasmid vector. Sample preparation flow chart for bacteria detection Collect cells by centrifugation or membrane filtration ↓ Lyse Cells using standard techiniques ↓ DNA extraction using standard techniques ↓ Analysis (Real-time PCR) Sample preparation flow chart for fungi (yeast and mold) detection Collect cells and cell fragments by centrifugation or membrane filtration ↓ Lyse Cells using standard techniques ↓ Extract DNA using standard techniques ↓ Analysis (Real-time PCR)

[0066] Isolation of DNA from Samples

[0067] DNA is isolated or extracted from the microorganism cells contained within the test sample. For example, DNA extraction may be performed using any of numerous commercially available kits for such purpose. One such kit, called IsoCode, is available from Schleicher and Schuell of Keene, N.H. The IsoCode kit contains paper filters onto which cells are applied. Through treatment of the paper filters as described by the manufacturer, most cellular components remain in the paper filter and DNA is released into an aqueous solution. The DNA in the solution can then be added to various enzymatic amplification reactions, as are discussed below.

[0068] Other commercially available kits exist for extraction of DNA from cells. Commercial kits do not have to be used, however, in order to obtain satisfactory DNA. Standard methods, well known to those skilled in the art, have been published in the scientific literature. Such methods commonly involve lysis of cells and removal of cellular components other than nucleic acids by precipitation or by extraction with organic solvents. Enzymatic treatment with proteases and ribonucleases can be used to remove proteins and RNA, respectively. DNA is then commonly precipitated from the sample using alcohol.

[0069] Real-Time PCR

[0070] A variety of methods can be used to determine if a PCR product has been produced. One way to determine if a PCR product has been produced in the reaction is to analyze a portion of the PCR reaction by agarose gel electrophoresis. For example, a horizontal agarose gel of from 0.6 to 2.0% agarose is made and a portion of the PCR reaction mixture is electrophoresed through the agarose gel. After electrophoresis, the gel is stained with ethidium bromide. PCR products are visible when the gel is viewed during illumination with ultraviolet light. By comparison to standardized size markers, it is determined if the PCR product is of the correct expected size.

[0071] The PCR procedure preferably is done in such a way that the amount of PCR products can be quantified. Such “quantitative PCR” procedures normally involve comparisons of the amount of PCR product produced in different PCR reactions. A number of such quantitative PCR procedures, and variations thereof, are well known to those skilled in the art. One inherent property of such procedures, however, is the ability to determine relative amounts of a sequence of interest within the template that is amplified in the PCR reaction.

[0072] One particularly preferred method of quantitative PCR used to quantify copy numbers of sequences within the PCR template is a modification of the standard PCR called “real-time PCR.” Real-time PCR utilizes a thermal cycler (i.e., an instrument that provides the temperature changes necessary for the PCR reaction to occur) that incorporates a fluorimeter (i.e. an instrument that measures fluorescence). In one type of real-time PCR, the reaction mixture also contains a reagent whose incorporation into a PCR product can be quantified and whose quantification is indicative of copy number of that sequence in the template. One such reagent is a fluorescent dye, called SYBR Green I (Molecular Probes, Inc.; Eugene, Oreg.) that preferentially binds double-stranded DNA and whose fluorescence is greatly enhanced by binding of double-stranded DNA. When a PCR reaction is performed in the presence of SYBR Green I, resulting DNA products bind SYBR Green I and fluoresce. The fluorescence is detected and quantified by the fluorimeter. Such technique is particularly useful for quantification of the amount of template in a PCR reaction.

[0073] A preferred variation of real-time PCR is TaqMan® (Applied Biosystems) PCR. The basis for this method is to continuously measure PCR product accumulation using a dual-labeled flourogenic oligonucleotide probe called a TaqMan® probe. The “probe” is added to and used in the PCR reaction in addition to the two primers. This probe is composed of a short (ca. 15-30 bases) oligodeoxynucleotide sequence that hybridizes to one of the strands that are made during the PCR reaction. That is, the oligonucleotide probe sequence is homologous to an internal target sequence present in the PCR amplicon. The probe is labeled or tagged with two different flourescent dyes. On the 5′ terminus is a “reporter dye” and on the 3′ terminus is a “quenching dye.” One reporter dye that is used is called 6-carboxy fluorescein (FAM). One quenching dye that is used is called 6-carboxy tetramethyl-rhodamine (TAMRA). When the probe is intact, energy transfer occurs between the two fluorochromes and emission from the reporter is quenched by the quencher, resulting in low, background fluorescence. During the extension phase of PCR, the probe is cleaved by the 5′ nuclease activity of Taq polymerase, thereby releasing the reporter from the oligonucleotide-quencher and producing an increase in reporter emission intensity. During the entire amplification process the light emission increases exponentially.

[0074] Because the detection in Taqman assay is based on complementary binding of the third oligonucleotide probe to the amplified PCR products, it can significantly minimize false positive results due to the detection of non-specific amplification and primer dimers in conventional PCR and other non-specific real-time PCR product detection approaches such as using SYBR Green or EtBr. However, the determination of proper primer and probe set needs more specified skills so that they will fit the product amplification and signal detection requirements.

[0075] Examples of primers and probes that are particularly useful in this procedure are listed above.

[0076] Fluorescence Detection

[0077] One example of an instrument that can be used to detect the fluorescence is an ABI Prism 7700, which uses fiber optic systems that connect to each well in a 96-well PCR tray format. The laser light source excites each well and a CCD camera measures the fluorescence spectrum and intensity from each well to generate real-time data during PCR amplification. The ABI 7700 Prism software examines the fluorescence intensity of reporter and quencher dyes and calculates the increase in normalized reporter emission intensity over the course of the amplification. The results are then plotted versus time, represented by cycle number, to produce a continuous measure of PCR amplification. To provide precise quantification of initial target in each PCR reaction, the amplification plot is examined at a point during the early log phase of product accumulation. This is accomplished by assigning a fluorescence threshold above background and determining the time point at which each sample's amplification plot reaches the threshold (defined as the threshold cycle number or CT). Differences in threshold cycle number are used to quantify the relative amount of PCR target contained within each tube.

[0078] Detecting Fungi in Samples

[0079] Oligonucleotide Primer and Probe Development for Detecting Yeast

[0080] We have cloned and sequenced the 18s rDNA gene fragments from representative yeast Zygosaccharomyces bailii (Lindner) Guilliermond strain ATCC 36947. We then compared our sequences against other published 18S rDNA sequences from molds, yeasts, and common eukarytic foods. We have also compared other target sequences including h1, h2, 23S rDNA, spacer sequence between 18S and 23S rDNA gene. We have developed primer-and-probe sequences that can detect the presence of generally all yeasts without cross-reacting with foods, molds or other bacteria. The aligned sequences of the 18S rDNA sequences of these yeast species are shown in FIG. 17. FIGS. 12, 13 and 14 show the full coding sequences for the genes corresponding to the alignments shown in FIG. 17.

[0081] Specificity Testing

[0082] Using the primer pair-and-probe set, all yeasts were tested positive in real-time PCR (FIGS. 15-19), while no cross-reaction was detected in other commonly found foodborne microorganisms and food items (FIGS. 15-19). Further specificity study revealed no combination of the above three oligonucleotides in other microorganisms after blast searching the nucleotide sequence database in the GenBank.

[0083] Oligonucleotide Primer and Probe Development for Detecting Mold

[0084] We have cloned and sequenced the 18s rDNA gene fragments of representative molds of food industry concerns, Byssochlamys fulva Olliver et Smith, teleomorph ATCC 24474 and Penicillium digitatum Saccardo, anamorph ATCC10030. Coloning primer up:TGCATGGCCGTTCTTAGTTGG(Z.B. code 64-75) (B.F. 667-688) (P.D. 674-695) down: GTGTGTACAAAGGGCAGGG(Z.B. 417-237) (B.F. 1011-1031) (P.D. 1029-1049). We then compared our sequences against other published 18S rDNA sequences from molds, yeasts, and common eukarytic foods. We have also compared other target sequences including h1, h2, 23S rDNA, spacer sequence between 18S and 23S rDNA gene. We have developed primer and probe sequences that can detect the presence of generally all mold without cross-reacting with foods, yeast or bacteria.

[0085] Specificity Test

[0086] Using primer pair-and-probe set, all yeasts were tested positive in real-time PCR (FIGS. 20 and 21), while no cross-reaction was detected in other commonly found foodborne microorganisms and food items (FIGS. 20 and 21). Further specificity study revealed no combination of the above three oligonucleotides in other microorganisms after blast searching the nucleotide sequence database in the GenBank.

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EXAMPLES Example 1

[0138] In this study, the 16s rDNA sequences of A. acidocaldarius, A. cycloheptanicus, and A. acidoterrestris were used as models for the development of specific primers and a flourogenic probe to be used in a real-time PCR assay. 16s rDNA was isolated from ATTC strains 43030, 49025, and 49029, then cloned into vectors, transformed into competent cells, and purified for sequencing. Following sequencing, the 16s rDNA sequences of the three strains were analyzed for the development of oligonucleotide primers and a flourescent probe. These primers and probe were used in a real-time PCR detection system where specificity and sensitivity tests were performed in media as well as beverage systems. This rapid detection system is unique because it can specifically detect not only the three original Alicyclobacillus species, but also detects newer species of Alicyclobacillus because of the genus-level 16s rDNA conservation of the priming sequences. This system can greatly benefit the food industry, particularly the beverage industry, by detecting the presence of Alicyclobacillus within hours, before the product ever reaches the consumer, saving not only time and money, but maintaining brand image and quality.

[0139] Materials and Methods

[0140] Bacterial strains and culture conditions. A. acidocaldarius strain ATTC 43030 was grown on ATCC 573 medium, consisting of 1.3 g (NH₄)₂SO₄, 0.37 g KH₂PO₄, 0.25 g MgSO₄.7H₂O, 0.07 g CaCl₂.2H₂O, 1.0 g glucose, 1.0 g yeast extract, and 1.0 L distilled H₂O. Solution pH was adjusted to 4.0 using H₂SO₄ and autoclaved at 121° C. for 15 minutes. A. acidoterrestris strain ATTC 49025 and A. cycloheptanicus strain ATCC 49029 were grown on BAM-SM ATCC 1656 medium consisting of 0.25 g CaCl₂.2H₂O, 0.5 g MgSO₄.7H₂O, 0.2 g (NH₄)₂SO₄, 3.0 g KH₂PO₄, 6.0 g yeast extract, 5.0 g glucose, 1.0 mL trace elements (0.66 g CaCl₂.2H₂O, 0.18 g ZnSO₄.7H₂O, 0.16 g CuSO₄.5H₂O, 0.15 g MnSO₄.4H₂O, 0.18 g CoCl₂.6H₂O, 0.10 g H₃BO₃, 0.30 g Na₂MoO₄.2H₂O, 1.0 L distilled H₂O), and 1.0 L distilled H₂O. Solution pH was adjusted to 4.0 using H₂SO₄ and autoclaved at 121° C. for 15 minutes. Stock cultures of all strains were stored in their respective media plus 40% glycerol and kept at −80° C.

[0141] Isolation of genomic DNA and amplification of 16s rDNA. DNA was isolated from 2% cultures of A. acidoterrestris strain ATTC 49025, A. cycloheptanicus strain ATCC 49029 A. acidocaldarius strain ATTC 43030 in respective media. Cultures were grown for 24 hours at 47° C. Genomic DNA was extracted from each strain using the Qiagen DNeasy Tissue Kit (Qiagen, Valencia, Calif.). The included protocol was followed, except the elution was repeated once with 100 μl of buffer AE. An approximately 1,500 bp region of the 16s rDNA was amplified from the genomic DNA using primers 8F and 1492R (15) with PCR performed on the Bio-Rad iCycler Thermal Cycler (Bio-Rad Laboratories, Hercules, Calif.). A 50 μl reaction mixture was used, containing 0.511 of primer 8F, 0.5 μl of primer 1492R, 1.0 μl of genomic DNA, 37 μl of sterile H₂O, 3 μl of 50 mM MgCl₂, 2 μl of a 10 mM dNTP mixture, and 1.01 Taq polymerase (Invitrogen, Carlsbad, Calif.). Amplification conditions included 30 cycles of 95° C. for 2 min, 42° C. for 30 s, and 72° C. for 4 min, with a final chain elongation for 20 min (15). PCR products were confirmed after 20 min of gel electrophoresis on 0.9% agarose gel at 100 volts, followed by 10 min of ethidium bromide staining for visualization.

[0142] Cloning and transformation of 16s rDNA gene. PCR products were purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). The protocol was followed as specified by the manufacturer, except 3011 of sterile H₂O was used in place of 50 μl of buffer EB for a single elution. Purified PCR products were then cloned into pCR 2.1 vectors using the TA Cloning kit (Invitrogen, Carlsbad, Calif.). A 10 μl ligation reaction for each PCR product was prepared as follows: 5 μl sterile H₂₀, 1 μl pCR 2.1 vector, and 2 μl PCR product were mixed together and incubated at 65° C. for 5 min, followed by 10 min of incubation on ice. 1 μl 10× ligation buffer and 1 μl T4 DNA ligase were then added to the mixture, followed by overnight incubation at 14° C. Transformation was then performed, beginning with centrifugation of the ligation reactions. Reactions were stored on ice while 50 μl of One Shot competent Escherichia coli cells were thawed for each transfer. 5 μl of each ligation reaction was added to a vial of One Shot cells and mixed gently, followed by incubation for 30 min on ice. Reactions were then heat shocked for 30 s at 42° C., and then placed on ice. 200 μl of SOC medium was added to each tube and then shook at 200 rpm for one hour at 37° C. The whole vial of cells was then spread onto LB agar plates containing X-Gal (20 mg/ml) and incubated at 37° C. overnight. Plates were stored at 4° C. following incubation.

[0143] Sequencing of 16s rDNA gene. Plates were observed for transformed (white) colonies. Five transformed colonies from each plate were selected using a sterile toothpick, then dipped into a microfuge tube containing 100 μl of sterile H₂O, and also spread on an LB agar plate. The stick was then placed into a tube containing 2 ml of LB broth and ampicillin (50 mg/ml). Plates were incubated at 37° C. overnight. LB tubes were shaken at 100 rpm at 37° C. overnight. Microfuge tubes were incubated at 100° C. for 10 min, followed by PCR to check for successful transformation. Standard 3-step PCR (CYCLES) was run with a 50 μl reaction mixture containing 0.5 μl of primer M13F, 0.5 μl of primer M13R, 1.0 μl of transformed DNA, 37 μl of sterile H₂O, 3 μl of 50 mM MgCl₂, 2 μl of a 10 mM dNTP mixture, and 1.0 μl Taq polymerase (Invitrogen, Carslbad, Calif.). PCR products were analyzed by gel electrophoresis. LB tubes were centrifuged for 10 min at 6000 rpm after overnight incubation and used in the QIAprep Spin Miniprep kit (Qiagen, Valencia, Calif.) following the manufacturer's protocol. 5 μl of product was set aside for PCR, and the rest of the miniprep yield was sent to be sequenced. Sequence data was entered into the NCBI BLAST network to search for similar sequences. Cloned sequences from ATCC strains 49025, 49029, and 43030 matched multiple 16s rDNA sequences from Alicyclobacillus species on the BLAST network.

[0144] Real-time Tagman PCR conditions. Fifty microliter reaction mixtures containing 0.5 μl of a 100 μM solution of CC16S-F primer, 0.5 μl of a 100 μM solution of CC16S-R primer, 0.5 μl of a 100 μM solution of CC16S-Probe, 33.3 μl of sterile H₂O, 5.0 μl of genomic DNA, 5 μl of 10× reaction buffer, 3 μl of MgCl₂, 2 μl of dNTP's, and 0.2 μl of Taq polymerase (Invitrogen, Carlsbad, Calif.) were used for specificity tests. For sensitivity assays, the following 50 μl reaction mixtures were used: 25 μl of 2× iQ Supermix, containing 100 mM KCl, 40 mM Tris-HCl, pH 8.4, 0.4 mM each dNTP, 50 U/ml iTaq DNA polymerase, 6 mM MgCl₂, and stabilizers (Bio-Rad, Hercules, Calif.), 0.5 μl of 100 μM stock CC16S-F primer, 0.5 μl of 100 μM stock CC16S-R primer, 0.5 μl of 100 μM stock CC16S-Probe, 5.0 μl of genomic DNA, and 18.5 μl of sterile H₂O. Real-time PCR was performed using the iCycler iQ Real-Time PCR Detection System (Bio-Rad, Hercules, Calif.). PCR conditions were as follows: 35-40 cycles of 95° C. denaturation for 30 s and 55° C. annealing for 30 s. The optical module was set to capture light during the annealing step. Results were analyzed using the iCycler iQ Optical System Software Version 3.0a (Bio-Rad, Hercules, Calif.).

[0145] Primer and probe design. Sequence alignments of the 16s rDNA sequences for strains 49025, 29029, and 43030 were constructed with ClustalV using MegAlign 5.01 (DNASTAR, Madison, Wis.). A sequence alignment of the 16S rDNA sequences was then performed for the following organisms: sequenced Alicyclobacillus strains ATCC 49025, 49029, and 43030, A. acidoterrestris strain DSM 3923 (AB042058), A. cycloheptanicus strain DSM 4006 (AB042059), A. acidocaldarius strain DSM 454 (AB059664), Geobacillus subterraneus strain K (AF276307), Sulfobacillus disulfidooxidans SD-11 (U34974), B. thermoleovorans strain ATCC 43513 (M77488), and Clostridium elmenteitii isolate E2SE1-B (AJ271453). The alignment was constructed with ClustalV using MegAlign 5.01 (DNASTAR, Madison, Wis.). Aligned regions were carefully scanned by eye to find areas of perfect identity within the representative Alicyclobacillus species in order to create PCR priming regions. The following criteria were used for primer and probe selection: (1) 100% identity between representative sequences, (2) priming region of less than 200 bp, (3) T_(m) greater than 55° C., (4) C or G in the terminal positions of both 5′ and 3′ ends, (5) greater than 45% C+G content, and (6) no visual hairpin loops or secondary structures, confirmed using the Oligo Toolkit (Qiagen, Valencia, Calif.) (22).

[0146] Specificity and sensitivity tests. Assays were performed using the aforementioned PCR conditions to test for specificity of the system for Alicyclobacillus spp. and any cross-reactions with other common food-borne microorganisms. Genomic DNA was extracted from broth cultures of 2% A. acidoterrestris, A. acidocaldarius, and A. cycloheptanicus grown for 48 h at 47° C. using the previously discussed DNA extraction protocol. In addition, genomic DNA was extracted from Escherichia coli DH-5α, Lactococcus lactis subsp. lactis, Geobacillus stearothermophilus ATCC 10149 and Pseudomonas putida 49L/51 to test specificity of the primers and probe.

[0147] Assays for the sensitivity of the real-time PCR assay for detection of Alicyclobacillus were performed using tenfold serial dilutions of 10⁰ to 10⁻⁸ of A. acidoterrestris in a 10 ml solution of 0.85% NaCl. Two percent cultures were initially grown for 48 h at 47° C. in order to obtain an OD₆₀₀ range between 0.400 and 0.800. After dilution, cells from 1 ml of each sample were collected by centrifugation at 12,000 rpm for 10 minutes for DNA extraction. Fifty microliter (50 μl) reaction mixtures containing 0.5 μl of CC16S-F primer, 0.5 μl CC16S-R primer, 0.5 μl CC16S-Probe, 33.3 μl of sterile H₂O, 5.0 μl of genomic DNA, 5 μl of 10× reaction buffer, 3 μl of MgCl₂, 2 μl of dNTP's, and 0.2 μl of Taq polymerase (Invitrogen, Carlsbad, Calif.) were used for each strain, as described above. Real-time PCR was carried out with the following cycling conditions: 35-40 cycles of 95° C. and 55° C., for 30 s each. After amplification, results were analyzed using the iCycler iQ Optical System Software Version 3.0a (Bio-Rad, Hercules, Calif.). A range of dilutions between 10⁻³ and 10⁻⁷ were plated on BBL Orange Serum Agar (Difco, Detroit) for colony counting. Plates were incubated at 47° C. for 48 h. Additionally, sensitivity tests were performed in the same manner using apple and orange juice. Also, 1 ml of culture was spiked in 9 ml of Powerade sports drinks and Minute Maid Lemonade to check for any inhibitory characteristics these drinks may display in a PCR assay.

[0148] Amplification, cloning, transformation, and sequencing of 16s rDNA gene. PCR was used to successfully amplify regions of 16s rDNA from A. acidoterrestrs, A. acidocaldarius, and A. cycloheptanicus using the 8F and 1492R primers. The Invitrogen TA cloning kit was used to insert the amplified 16s rDNA segment of each strain into pCR 2.1 vectors, and subsequently transformed into E. coli competent cells. Purified samples were then sent to the Plant-Microbe Genomic Facility at the Ohio State University and sequenced using an ABI PRISM 3700 DNA Analyzer (Applied Biosystems, Foster City, Calif.). TABLE V Oligonucleotide data for Alicyclobacillus spp. CC16S probe and primers. Name Sequence Length T_(m) G + C content CG16S-F CGTAGTTCGGATTGCAGGC 19 bp 65.6° C. 57.9% CC16S-R GTGTTGCCGACTCTCGTG 18 bp 63.3° C. 61.1% CC16S-Probe CGGAATTGCTAGTAATCGC 19 bp 57.9° C. 47.4%

[0149] Development of CC16S primers and probe. Sequence data obtained from the Plant-Microbe Genomics Facility was compiled and entered into the NCBI BLAST network to check sequence integrity. Sequence data for each strain corroborated with respective sequence data in the GenBank. The 16S rDNA sequences from the three sequenced strains, as well as from A. acidoterrestris strain DSM 3923 (AB042058), A. cycloheptanicus strain DSM 4006 (AB042059), and A. acidocaldarius strain DSM 454 (AB059664) were used as positive controls in the alignment to determine a suitable priming region. B. thermoleovorans strain ATCC 43513 (M77488) and Clostridium elmenteitii isolate E2SE1-B (AJ271453) were used as negative controls in the alignment. In addition, closely related Geobacillus subterraneus strain K (AF276307) and Sulfobacillus disulfidooxidans SD-11 (U34974) were added to the alignment. Using the criteria described in the methodology, a forward and reverse primer and fluorogenic probe were derived, named CC16S-F, CC16S-R, and CC16S-Probe respectively. The sequences for the oligonucleotides are shown in Table V. This oligonucleotide set will amplify a 134 bp segment of the 16S rDNA. The alignment of the 134 bp priming region is shown in FIG. 22, with the selected primer and probe oligonucleotide sequences boxed around the Alicyclobacillus strains. These sequences were entered into the BLAST search network in order to discover identities with other unrelated organisms to ensure their specificity for Alicyclobacillus. Results show that the priming sequences are specific for 16S rDNA sequences of the three Alicyclobacillus species sequenced. In addition, the priming sequences also match the newly discovered species A. hesperidum, A. herbarius, A. acidiphilus, and A. sendaiensis. Also, it was found after alignment and BLAST searches that the priming region was highly similar to the members of the Geobacillus and Sulfobacillus genera, two closely related groups. Primers CC16S-F and CC16S-R were ordered from Sigma-Genosys (The Woodlands, Tex.), and the CC16S-Probe was ordered from Biosearch Technologies (Novato, Calif.). CC16S-Probe was labeled with the reporter dye Quasar 670 on the 5′ end, and quencher dye BHQ-2 on the 3′ end.

[0150] Real-time PCR specificity assay. Real-Time PCR is a new method has been developed to overcome the problems of standard PCR while increasing sensitivity and allowing for nearly instantaneous results. Real-time PCR adds an optical module and a fluorogenic probe to a standard PCR assay, while including computer-based data analysis software for real-time monitoring. Real-time PCR eliminates the need for post-amplification analysis and is not affected by non-specific amplification. The optical module attached to the thermal cycler detects a flourescent signal that is emitted from the labeled probe at each cycle during the annealing stage. The amount of emission is recorded by computer software and plotted as an exponential curve, displaying the cycle at which a significant amount of amplification takes place.

[0151] The flourescent reporter dye is held on the 5′ end of an oligonucleotide probe, with a quenching dye on the 3′ end to capture flourescence not related to amplification. When the probe anneals within the primed region, the 5′ exonuclease activity of the polymerase in the reaction system cleaves the probe, inhibiting the quencher dye and increasing the emitted flourescence from the 5′ reporter dye (21).

[0152] A real-time PCR assay was developed to test the specificity of the primers and probe for A. acidoterrestris, A. acidocaldarius, and A. cycloheptanicus. The assay also included E. coli DH-5α, L. lactis subsp. lactis, and P. putida to test for any unwanted cross-reactions with common foodborne microorganisms. In addition, Geobacillus stearothermophilus ATCC 10149 was included in the assay since it is a closely related thermophile of the Bacillus subfamilies. Assays were performed in triplicate, and results analyzed using the iCycler iQ Optical System Software. The results show that the reaction is specific for the three Alicyclobacillus while not reacting with E. coli DH-5α, L. lactis subsp. lactis, or P. putida. However, G. stearothermophilus had a positive reaction within the system.

[0153] Real-time PCR sensitivity assay and limit of detection. After establishing system specificity, sensitivity of detection was determined. In order to accomplish this, tenfold serial dilutions in a 0.85% NaCl solution were made using A. acidoterrestris ATCC 49025 cultures. Real-time PCR assays were run in triplicate and results were analyzed using the iCycler iQ Optical System Software. A typical result is shown in FIG. 23. Quantification of the lowest detection level was performed through colony counting of plated dilutions used in the PCR. Colonies were counted on OSA plates and then averaged. The CFU/ml was calculated, and cell counts were determined for the lowest positive curve by multiplying the CFU/ml by the dilution factor of the curve. Data for cell counts and detection limits is presented in Table VI. In FIG. 24, the lowest accurate curve presented is from a 10⁻⁵ dilution, which is equivalent to 160 CFU/ml by plate count. Sensitivity tests were performed in triplicate, with the limit of detection ranging between 66 and 160 cells. The mean detection limit is 103 cells. TABLE VI A. acidoterrestris cell counts and corresponding detection limits for sensitivity tests performed in saline solution and orange juice. Mean cell count Minimum PCR Mean PCR Mean number per replicate detection level detection level Replicate Media of colonies^(a) (CFU/ml) per replicate^(c) for trial set^(d) 1 Saline 8  8.3 × 10^(6b)  8.3 × 10¹ Saline solution 2 Saline 160 1.60 × 10⁷ 1.60 × 10² 1.03 × 10² 3 Saline 66  6.6 × 10⁶  6.6 × 10¹ 1 Orange Juice 21  2.1 × 10⁷  2.1 × 10¹ Orange juice 2 Orange Juice 63  6.3 × 10⁷  6.3 × 10¹ 5.36 × 10¹ 3 Orange Juice 76  7.6 × 10⁷  7.6 × 10¹

[0154] The detection limit of the Alicyclobacillus real-time PCR rapid screening system was also established in beverages using orange juice as a diluent. Serial dilutions were performed as previously described with juice in place of 0.85% NaCl. Juice samples were initially run in parallel with samples in 0.85% NaCl, and CT values and curve intensities were found to be comparable in both systems. Results for the assay in orange juice are shown in FIG. 25. Colony counting was performed on plated dilutions used in the PCR in order to determine cell counts at the minimum detection level. Data for cell counts and detection limits is presented in Table VI. In FIG. 25, the lowest accurate curve presented is from a 10⁻⁶ dilution, which is equivalent to 63 CFU/ml. Sensitivity tests were performed in triplicate, with the limit of detection ranging between 21 and 76 cells. The mean detection limit is 54 cells.

[0155] The efficiency of the system has also been tested in other beverages including apple juice, three sports drinks and Lemonade purchased from local grocery stores. These beverages were spiked with A. acidoterrestris cultures followed by cell collection, DNA extraction and real-time PCR detection. In all these cases, expected PCR amplification results were obtained indicating no particular inhibition by the ingredients from these tested beverages.

[0156] Discussion

[0157] A specific and sensitive real-time PCR-based rapid detection system for Alicyclobacillus has been developed. In the past, PCR based assays have been used to detect microorganisms in different environments (16, 2, 17, 18, 19, 20, 28). More recently, the use of real-time PCR has been a favorable alternative to standard PCR based assays due to the increased speed and sensitivity of the results, the ability to quantify detection levels, and the elimination of post-amplification analysis (21). The present method was developed by targeting the 16s rDNA gene of Alicyclobacilli, using A. acidoterrestris, A. acidocaldarius, and A. cycloheptanicus as models for primer and probe development. However, the developed primers and probe could also be beneficial in detecting newly classified members of Alicyclobacillus, due to high sequence identity as shown by the BLAST data. This real-time PCR assay is an improvement over traditional culture methods of detection and PCR based detection systems. Culture methods can take between three and seven days for results to be available (12, 13). While accurate, the time frame is much too long for practical industry implementation. PCR assays provide much quicker results, but false positives can be easily detected (21), and gel electrophoresis analysis must be performed after amplification. Real-time PCR assays can be readily implemented in the industry because of the real-time results. Samples can be taken from the floor as they are produced and the presence of Alicyclobacilli can be detected within 3 hours.

[0158] In this study, the developed primers and probes were able to specifically detect A. acidoterrestris, A. acidocaldarius, and A. cycloheptanicus without cross-reaction with other common foodborne microorganisms. In addition, the system could also detect the presence of G. stearothermophilus.

Example 2

[0159] A real-time PCR based rapid system was developed for detecting spoilage Alicyclobacillus spp. in foods. A common gene of Alicyclobacillus spp. encoding squalene-hopene cyclase, a key enzyme involved in hopanoid biosynthesis, was targeted for specific primers and probe development. Using the combination of the primers and probe, specific detection of the presence of representative strains from Alicyclobacillus spp. was achieved in the Taqman-based real-time PCR assay without cross-reacting with other food-borne bacteria. The presence of around 100 cells in collected samples can be detected within several hours.

[0160] Food spoilage causes significant financial loss to the industry. Every year, about 10% of our food supplies are lost due to spoilage and a significant portion of the problem is because of the presence of spoilage microbial agents, particularly molds, yeasts, and bacteria capable of surviving moderate heat- and acidic-treatments. Due to the limitation of applying extreme processing conditions, which can significantly alter the physiochemical properties and nutritional values of many food products, proper detection screening for the presence of microbial spoilage agents in food becomes a prior choice for quality control in the food industry. However, conventional industry practices for microbial detection from plate counting to biochemical analysis take anywhere from 48 hours to a couple of weeks. These methods are especially unsuitable for products with limited shelf life such as fruit juices. Novel detection approaches enabling rapid and specific detection of spoilage microorganisms within hours are preferred.

[0161] While the polymerase chain reaction (PCR) has been used extensively for years to rapidly amplify targeted DNA sequence regions, certain shortcomings limit its application in diagnostics and detection. For instance, PCR product analysis must be carried out after amplification, giving rise to an issue of post-amplification contamination and carry-over contamination (Heid et al., 1996). Most importantly, a high ratio of false positive results are often associated with PCR due to non-specific binding of the primers and the subsequent non-specific amplification of products. Recently a real-time PCR technology has emerged as a powerful diagnostic tool in both medical and agricultural fields.

[0162] Using real-time PCR, a fluorescent dye such as SYBR green can be incorporated into the reaction mixture and the fluorescent signals, generated from fluorescent dye binding to double stranded DNA products, can be detected directly by the optical module coupled with the thermocycler. The signals are processed by computer data analysis software for almost real-time calculation and on screen plotting. A new dimension of real-time PCR called Taqman assay further introduced a third oligonucleotide probe, labeled with 5′ fluorescent reporter dye and 3′ quenching dye, for signal detection (Livak et al., 1995; Basseler et al., 1995). In the Taqman system, the quenching dye on the 3′ end captures the fluorescence from the 5′ reporter dye so the intact probe itself does not produce strong signal. During amplification when the probe hybridized to complementary sequence within the amplified products, the 5′→3′ exonuclease activity of the polymerase in the reaction system cleaves the probe, minimized the quenching effect and the emitted fluorescent signal from the 5′ reporter dye can be detected by the optical module. An advantage of applying the Taqman system is that a double complementing sequence selection mechanism by both the primers and the probe is involved, therefore the false positive rate of the detection can be significantly cut down. So far, various Taqman real-time PCR-based detection approaches have been reported. However, reports on its application in the real food system are still limited. The greatest challenges are (i) effective extraction of DNA and RNA from a system where microorganisms are mixed with the food matrix including bulk proteins, carbohydrates and fatty acids, (ii) selection of primer-and-probe sets that are specific for the target microorgnanisms and do not interaction with background microflora and food ingredients, and (iii) minimizing the influence of food ingredients and other chemical compounds in the food matrix on the action of enzymes involved in DNA extraction and amplification.

[0163] Our objective was to demonstrate the feasibility of the real-time PCR based detection technology for food industry applications. It is our understanding that due to the complication of various food systems, detection procedures likely need to be optimized for individual food commodities. In this study, we investigated the practicability of using the Taqman-based real-time PCR approach in detecting target microorganisms in juice products. Here we report the effectiveness of the Taqman-based detection system in rapid, specific and sensitive detection of spoilage A. acidocaldarius and A. acidoterrestris in juice products, using a primer-and-probe set specific for the shc gene encoding squalene-hopene cyclase.

[0164] Materials and Methods

[0165] Bacterial Strains and Growth Conditions.

[0166] The bacterial strains used in the study and their growth conditions were listed in Table VI. ATCC 573 medium consists of 1.3 g (NH₄)₂SO₄, 0.37 g KH₂PO₄, 0.25 g MgSO₄.7H₂O, 0.07 g CaCl₂.2H₂O, 1.0 g glucose, 1.0 g yeast extract, and 1.0 L distilled H₂O, pH 4.0. BAM-SM ATCC 1656 medium consists of 0.25 g CaCl₂.2H₂O, 0.5 g MgSO₄.7H₂O, 0.2 g (NH₄)₂SO₄, 3.0 g KH₂PO₄, 6.0 g yeast extract, 5.0 g glucose, 1.0 mL trace elements (0.66 g CaCl₂.2H₂O, 0.18 g ZnSO₄.7H₂O, 0.16 g CuSO₄.5H₂O, 0.15 g MnSO₄.4H₂O, 0.18 g CoCl₂.6H₂O, 0.10 g H₃BO₃, 0.30 g Na₂MoO₄.2H₂O, 1.0 L distilled H₂O), and 1.0 L distilled H₂O. Geobacillus stearothermophilus ATCC 10149 was grown in Nutrient broth (Difco). Stock cultures of all strains were stored in their respective media plus 40% glycerol and kept at −80° C. All inoculations used were 2% concentrations made from frozen cultures. TABLE VII Bacteria cultures used in the study. Medium Strains and Growth Condition Resource A. acidocaldarius ATCC43030 #573 broth^(a) at 48° C. ATCC A. acidoterrestris ATCC49025 #1655 broth^(a) at 48° C. ATCC A. cycloheptanicus ATCC49029 #1656 broth^(a) at 48° C. ATCC Bacillus subtilis Nutrient broth^(b), 40° C. Geobacillus? E. coli DH5α LB broth, Miller^(c) at 37° C. Pseudomonus putidis? LB broth, Miller at 37° C. Listeria monocytogenes V7 Tryptic soy broth^(d) at 37° C. Lactococcus lactis 2301 M17 broth^(e) at 37° C.

[0167] DNA extraction, gene cloning and DNA sequencing. For DNA extraction, cells were collected from 1 ml of bacterial culture by micro-centrifugation 7.6K rpm for 10 min. The cell pellet was treated with 20 mg/ml of lysozyme (Sigma Chemical CO. St Louis, Mo. 63178, USA) in buffer for 45 min at 37° C. Genomic DNA was extracted using the DNeasy® Tissue Kit (QIAGEN GmbH, D-40734 Hilden, Germany) and eluted into 100 μl of elution buffer following the instructions from the manufacturer.

[0168] The shc gene fragment from each strain was obtained by conventional PCR amplification using degenerate primers derived from conserved amino acid sequences and the genomic DNA from each strain as template. The reaction mixture includes 1×PCR buffer, 3 mM MgCl₂, 4 mM dNTP (Invitrogen, Carlsbad, Calif.), 1 μM primer pairs, 1 μl of genomic DNA template and ddH₂O in a total final volume of 50 μl. PCR was performed one cycle at 95° C. for 3 min, followed by 30 cycles at 95° C. for 30 s, 50° C. for 30 s and 72° C. for 1 min, with a final extension at 72° C. for 7 min using I-cycler (Bio-Rad, Hercules, Calif.). PCR products were purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.) following manufacturer's instruction. Purified PCR products were cloned into pCR 2.1 vectors and transformed into One Shot competent Escherichia coli cells using the TA Cloning kit (Invitrogen, Carlsbad, Calif.). Recombinant plasmids were recovered using QIAGEN miniprep (QIAGEN GmbH, D-40734 Hilden, Germany). DNA sequences were determined using the ABI PRISM® 3700 DNA Analyzer (Applied Biosystems, Foster City, Calif.) at the Plant Genome Sequence Facility, The Ohio State University.

[0169] Real-time Tagman PCR conditions For real-time PCR, the reaction was conducted in thin-wall microcentrifuge tubes including 1× iQ™ Supermix (Bio-Rad, Hercules, Calif.), 0.5 μM of primer pair, 0.3 μM of probe, 10 μl of genomic DNA extraction and ddH₂O in a final volume of 50 μl. PCR was performed one cycle at 95° C. for 3 min followed by 40 cycles of 95° C. for 30 s, 55° C. for 1 min using I-cycler (Bio-Rad, Hercules, Calif.).

[0170] DNA sequence analysis. The DNASTAR (DNASTAR, Madison, Wis.) software package was used in DNA and protein sequence alignment and homology search. DNA oligonucleotide primer and probe sequences were also compared with sequences from the GenBank sequence database using BlastSearch.

[0171] Specificity and sensitivity analyses Assays were conducted to test the specificity of the detection system against spoilage Alicyclobacillus spp. and other common food-borne microorganisms. Genomic DNA was extracted from broth cultures of A. acidoterrestris and A. acidocaldarius, grown for 48 h at 48° C. (absorbance at OD₆₀₀ around 0.5-0.7), using the previously discussed DNA extraction protocol. Genomic DNAs extracted from 1 ml of overnight culture of Escherichia coli DH-5α, Lactococcus lactis subsp. lactis C2, Geobacillus stearothermophilus ATCC 10149 and Pseudomonas putida 49L/51 were also used in the specificity study. Ten micro liters out of the 100 micro liter of elution was used as template and the real-time PCR amplification was carried out using conditions described above but using 32 instead of 40 cycles of amplification.

[0172] The sensitivity tests of the real-time PCR assay for detection of Alicyclobacillus in bacterial culture media were performed using tenfold serial dilutions from 10⁰ to 10⁻⁸ of A. acidoterrestris in a 10 ml solution of 0.85% NaCl. The initial cultures were obtained by grown for 18 h at 48° C. using 2% inoculation from the frozen stock, with the absorbance reading at OD₆₀₀ range between 0.38 and 0.42. After serial dilution, cells from 1 ml of each sample were collected by centrifugation at 7600 rpm for 10 minutes for DNA extraction. Ten microliter out of the 100 microliter of elution was used as template and the real-time PCR amplification was carried out as described above.

[0173] Sensitivity tests in juice products were also performed in the same manner but the serial dilutions were carried in apple juice instead of saline.

[0174] In both sensitivity analyses, a range of dilutions between 10⁻⁴ and 10⁻⁵ were plated on acidified PDA agar (Difco, Detroit) for colony counting to compare with the results by Taqman real-time PCR. Plates were incubated at 48° C. for 48 h.

[0175] Results

[0176] 1. The Primer-and-Probe Set Used in the Real-Time PCR Taqman Assay.

[0177] Hopanoids are membrane components involved in maintaining membrane fluidity and stability (4) of Alicyclobacillus spp. in extreme environmental conditions. We have targeted the shc gene encoding squalene-hopene cyclase, a key enzyme in hopanoid biosynthesis, for PCR primer-and-probe development.

[0178] Using an established approach (Wang et al., 2001), squalene-hopene cyclase protein sequences from several microorganisms were aligned and conserved amino acid sequences in squalene-hopene cyclase were identified. FIGS. 5 and 6, respectively, show the polynucleotide and protein alignments for two strains of Alicyclobacillus. Two degenerate primers 5′ GGNGGNTGGATGTTYCARGC 3′ (Y=C+T; R=A+G; N=A+T+C+G) (SEQ ID NO 64) and 5′ YTCNCCCCANCCNCCRTC 3′ (SEQ ID NO 65) were derived. Using this set of primers and the genomic DNA from A. acidocaldarius ATCC 43030 and A. acidoterrestris ATCC 49025, the 705 bp shc fragments were amplified by PCR from both strains. The PCR fragments were cloned into the TA vector and the inserted DNA sequences were determined. The DNA sequences were further compared with other Alicyclobacillus spp. shc sequences in the GenBank. Three conserved oligonucleotides were derived including the Forward Primer 5′ ATGCAGAGYTCGAACG 3′ (SEQ ID NO 25) and the Reverse Primer 5′ AAGCTGCCGAARCACTC 3′ (SEQ ID NO 27) flanking a 136 bp fragment, and the Probe 5′TCRGARGACGTCACCGC3′ (SEQ ID NO 26). The synthesized primers were ordered from Sigma-Genosys (The Woodlands, Tex.). The Probe is fluorescence-labeled with 5′ 6-FAM BHQ-1 3′ by Biosearch Technologies, Inc. (Novato, Calif.) and was used in the Taqman assay.

[0179] Specific Detection of Spoilage Alicyclobacillus spp.

[0180] Real-time PCR assays were performed to determine the specificity of the primers and probe for spoilage Alicyclobacillus spp. E. coli DH-5α, L. lactis subsp. lactis C2, and P. putida 49L/51, G. stearothermophilus ATCC 10149 were also included in the study to test the possibility of cross-reactions by the primer-and-probe set with common food-borne microorganisms. Assays were performed in triplicate, and a representative real-time PCR curve plotted by the iCycler iQ Optical System Software is shown in FIG. 26.

[0181] Representative strains from A. acidocaldarius and A. acidoterrestris were tested positive. No cross-reaction was detected in other commonly found food-borne microorganisms. Further specificity study was conducted by searching the Blast databases for DNA sequences from the National Center for Biotechnology Information (NCBI). We found no combination of the above three oligonucleotides in other microorganisms but A. acidocaldarius and A. acidoterrestris. The data suggested that the system is specific for spoilage A. acidocaldarius and A. acidoterrestris.

[0182] Levels of Detection in Bacterial Culture Medium and in Apple Juice.

[0183] To establish the detection level using the above real-time PCR system, we have conducted 10⁰ to 10⁻⁶ serial dilutions of A. acidoterrestris ATCC 49025 in culture medium. Cells from 1 ml of diluted samples were collected and {fraction (10/100)} of the DNAs extracted were used as template in the real-time PCR analysis. All experiments were repeated for at least three times and a representative curve was presented as FIG. 27. Our results showed that using the above primer-and-probe set, the presence of as few as 10 cells cells in a sample could be detected. This detection level is comparable to results from other microbial detection studies using real-time PCR.

[0184] To further verify the feasibility of using the detection system in juice products, we have conducted 10⁰ to 10⁻⁶ serial dilutions of A. acidoterrestris ATCC 49025 in apple juice. The experiments were repeated for three times and a representative curve was presented as FIG. 28. Similar detection level was achieved in apple juice.

[0185] 2. Discussion and Conclusion

[0186] Rapid, specific and sensitive detection of microorganisms in agricultural and food systems has proved to be a challenge. There are several major hurdles for effective microbial detection in the food systems. First, problematic food is normally associated with low level of initial contamination. However, the rich food matrix can support the growth of microbial agents in many cases during food storage and distribution. Thus even low level of initial contamination can cause serious damage. To be able to detect the presence of this low level contamination from food matrix often involving bulk proteins, carbohydrates and fatty acids, proper sampling and lengthy pre-detection enrichment steps are often required. To achieve rapid detection, pre-detection enrichment procedures need to be minimized and the detection system also should be sensitive enough to pick up low level of contamination.

[0187] Second, both foods and farm environment are complex ecosystems with significant background microflora. In addition to the background microflora normally associated with raw materials, beneficial microorganisms such as starter cultures sometimes are intentionally inoculated and present in large quantity in certain products. Therefore, to avoid false positive results, detection method for spoilage or pathogenic organisms needs to be specific enough to pick up only the target microorganisms. Finally, the rich and complex food ingredients often include various salts, carbohydrates, preservatives, emulsifiers, fatty acids, and proteins. The presence of these components varies among food commodities and can interfere with detection in various degrees. Therefore detection approaches and procedures need to be verified for effectiveness in these food systems.

[0188] Real-time Taqman PCR-based approach has the potential to achieve rapid, sensitive and specific detection. An average DNA amplification cycle for a small fragment can be completed within a minute. Theoretically after 30-40 cycles the amplification products from one DNA template in the system can be readily detected and plotted on the screen in almost real-time. The double sequence selection mechanism involving both the oligonucleotide primers and probe further minimizes the possibility of false positive results and enhances the detection specificity.

[0189] In this study, using a primer-and-probe set targeting the spoilage A. acidocaldarius and A. acidoterrestris, we were able to achieve specific detection without cross-reacting with representative strains from other common food-borne microorganisms including a strain from the closely related thermophilic G. stearothermophilus. Although only a few representative strains were used in the laboratory specificity studies, a computer-based search covering all the world-wide deposited DNA sequences available through the NCBI website was conducted to ensure that the combination of the sequences of the oligonucleotide primers and probe used in the study are distinctive enough to detect only A. acidocaldarius and A. acidoterrestris strains.

[0190] The level of detection limit with confidence is important for any detection approaches. In this study we have conducted sensitivity tests in both bacterial cultural medium and a real food system-apple juice. For laboratory handling purpose and for the convenient of using commercially available yet economically feasible DNA extraction kit, bacterial cells were serially diluted in either medium or juice and cells in 1 ml of samples were collected by micro-centrifugation. DNA were extracted and {fraction (10/100)} of the elution were used as template in PCR. The experiment was repeated at least three times and a representative curve presented as FIG. 27. The lowest detection limit was determined based on the cell count numbers from agar plates derived from dilution with the optimal counting numbers (30-300) and the fold of dilution corresponding to each positive curves presented. Using this approach, we report that the presence of as few as 10 cells per sample with confidence. Because during each independent repeats the 10-fold serial dilutions were conducted without knowing exactly how many cells were in 1 ml of samples, the standard deviation reflects this fact. To further narrow down the range of standard deviation of detection, serial dilutions within the range of 2-10 can be conducted so a more precise confident level can be possibly established. We did not extrapolate the results using In other referred paper sometimes a standard curve was established first for sensitivity analysis. Furthermore, in a quality control laboratory, a regular sample size is normally 25 ml instead of 1 ml. Theoretically, sample detection limits can further be improved as long as cells from 25 ml or even 100 ml of samples can be properly collected and re-suspended in 1 ml of solution to conduct DNA extraction.

[0191] We are in the process of establishing a rapid detection system for food industry applications (the CleanPlant system) and the real-time Taqman PCR is one of our preferred platforms. In order to apply this detection platform in juice related products, we need to establish the feasibility of using the system for raw material screening and final product monitoring. We have conducted the sensitivity test by spiking the Alicyclobacillus in apple juice purchased from local grocery stores and similar level of detection was achieved indicating the applicability of such a system in final product screening. Further, we have used this system to detect the presence of Alicyclobacillus in apple juice concentrates, which are considered raw materials for the processing facilities. Similar level of detection was achieved except diluting and rinsing procedures need to be incorporated to minimize inhibitory effects by the concentrated food ingredients (data not shown). These data suggested that

[0192] Because the system we developed is based on recognition of the signature DNA sequence of microorganisms, it has high specificity and does not cross react with other food-borne microorganisms (FIG. 26). The detection limit was achieved in both bacterial culture medium and apple juice. Since no inhibition to the reaction system was detected using samples collected from apple juice, we expect the sensitivity of the detection system can be further improved by including a pre-treatment procedure to apply a centrifugation or membrane filtration procedure to concentrate the bacteria cells from a large sample volume. This approach is in fact a preferred practice in the industry where the sampling size varies from 25 ml to 1 liter. Since only {fraction (1/10)} of the DNA extract was used in the reaction, we expect further improvement for the sensitivity can be achieved by incorporating more DNA template to the reaction system.

Example 3

[0193] Yeast Genomic DNA Extraction Protocol

[0194] Innoculate yeast, overnight; Centrifuge 10,000 rpm for 10 mins; Discard supernatant, add 600 ul Sorbital buffer (1 M Sorbital, 100 mM EDTA, 14 mM B-mercaptoethanol, 30 ul 20 mg/ml lyticase) in pellet, vortex, room temperature for 30 min; Centirfuge 10,000 rpm for 5 min; Add 180 ATL (Qiagen DNAeasy kit) and 20 ul proteinase K (Qiagen DNAeasy kit) to pellet and vortex; 55°. for 1 h, add 200 ul AL (Qiagen DNAeasy kit), 70°. for 10 min; 200 ul Ethanol, vortex, apply to DNeasy spin column; centrifuge 10,000 rpm for 1 min, discard flow-through, add 500 ul Buffer AW1 (Qiagen DNAeasy kit), spin for 1 min; add 500 ul Buffer AW1 (Qiagen DNAeasy kit), spin for 3 min; add 100 ul AE buffer (Qiagen DNAeasy kit), spin for 1 min.

[0195] Mold Genomic DNA Extraction Protocol:

[0196] Innoculate Mold in PDB; 3 days later, centrifuge 10,000 rpm for 10 min; add 500 ul Mold extraction buffer (1% CTAB, 1.4 M NaCl, 100 mM Tris, 20 mM EDTA, pH 8.0) to pellet; 100 ul glass beads, water bath sonic (55°.) for 45 min; add 50 ul Proteinase K (Qiagen DNAeasy kit) and incubate in 55°. for 1 h; Centrifuge 10,000 rpm for 5 min; Transfer the supernatant, add 500 ul AL (Qiagen DNAeasy kit), 70°. for 10 min; Add 200 ul Ethanol and pipet it into Dneasy mini column; 10,000 rpm for 1 min; Add 500 ul AW1 (Qiagen DNAeasy kit), spin for 1 min; Add 500 ul AW2 (Qiagen DNAeasy kit), spin for 3 min; Add 100 AE buffer (Qiagen DNAeasy kit), spin for 1 min.

1 140 1 19 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 1 gagcccgcgg cgcattagc 19 2 17 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 2 gcgacgatgc gtagccg 17 3 16 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 3 cgcaatgggc gcaagc 16 4 16 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 4 gcttgcgccc attgcg 16 5 19 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 5 gagcaacgcc gcgtgagcg 19 6 16 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 6 cttcgggttg taaagc 16 7 15 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 7 cggctaacta cgtgc 15 8 15 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 8 gcacgtagtt agccg 15 9 18 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 9 agtgctggag aggcaagg 18 10 17 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 10 ctggacagtg actgacg 17 11 20 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 11 gcacgaaagc gtggggagca 20 12 20 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 12 tgctccccac gctttcgtgc 20 13 20 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 13 ggagtacggt cgcaagactg 20 14 17 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 14 cgcacaagca gtggagc 17 15 14 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 15 cagggcttga catc 14 16 14 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 16 gatgtcaagc cctg 14 17 19 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 17 ggcgtaagtc ggaggaagg 19 18 19 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 18 atgtcctggg ctacacacg 19 19 19 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 19 gcctgcaatc cgaactacc 19 20 19 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 20 cgtagttcgg attgcaggc 19 21 20 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 21 cggaattgct agtaatcgcg 20 22 18 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 22 cacgagagtc ggcaacac 18 23 18 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 23 gtgttgccga ctctcgtg 18 24 16 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 24 gatgattggg gtgaag 16 25 16 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 25 atgcagagyt cgaacg 16 26 19 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 26 tcgagaagga cgtcaccgc 19 27 17 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 27 aagctgccga arcactc 17 28 17 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 28 tactggtggg ggccgct 17 29 17 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 29 tactggtggg cgccgct 17 30 19 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 30 atggaagcgg agtacgtcc 19 31 20 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 31 atggaagcgg agtacgtcct 20 32 19 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 32 atggaagcgg aatatgtgc 19 33 20 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 33 atggaagcgg aatatgtgct 20 34 14 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 34 cgcgaggacg gcac 14 35 18 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 35 cgcgaggacg gcacgtgg 18 36 14 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 36 cgcgaagacg gcac 14 37 18 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 37 cgcgaagacg gcacctgg 18 38 17 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 38 caaaaggcgc tcgactg 17 39 18 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 39 caaaaggcgc tcgactgg 18 40 22 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 40 caaaaggcgc tcgactgggt cg 22 41 17 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 41 caaaagtcgc tcgactg 17 42 18 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 42 caaaagtcgc tcgactgg 18 43 22 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 43 caaaagtcgc tcgactggct cg 22 44 18 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 44 ggacggcggc tggggcga 18 45 21 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 45 ggacggcggc tggggcgagg a 21 46 27 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 46 ggacggcggc tggggcgagg actgccg 27 47 18 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 47 ggatggcggt tggggtga 18 48 21 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 48 ggatggcggt tggggtgaag a 21 49 27 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 49 ggatggcggt tggggtgaag attgccg 27 50 16 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 50 tgatggcgct catcgc 16 51 23 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 51 tgatggcgct catcgcgggc ggc 23 52 25 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 52 accccgtcgc agacggcctg ggcgc 25 53 25 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 53 acaccgtcgc agaccgcctg ggcgt 25 54 19 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 54 gtggtgctag catttgctg 19 55 18 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 55 gttagactcg ctggctcc 18 56 23 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 56 tttcaagccg atggaagttt gas 23 57 21 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 57 cggtttcaag ccgatggaag t 21 58 30 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 58 cctactaaat agggtgctag catttgctgg 30 59 26 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 59 ctaaataggg tgctagcatt tgctgg 26 60 25 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 60 cggtttcaag ccgatggaag tttga 25 61 17 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 61 ccgctggctt cttaggg 17 62 19 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 62 agggccagcg agtacatca 19 63 20 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 63 ctcaagccga tggaagtgcg 20 64 20 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 64 ggnggntgga tgttycargc 20 65 18 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 65 ytcnccccan ccnccrtc 18 66 19 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 66 cgtagttcgg attgcaggc 19 67 19 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 67 cggaattgct agtaatcgc 19 68 18 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 68 cacgagagtc ggcaacac 18 69 21 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 69 tgcatggccg ttcttagttg g 21 70 19 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 70 gtgtgtacaa agggcaggg 19 71 19 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 71 cgtagttcgg attgcaggc 19 72 18 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 72 gtgttgccga ctctcgtg 18 73 19 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 73 cggaattgct agtaatcgc 19 74 1500 DNA Alicyclobacillus acidocaldarius 74 agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60 gggtctcttc ggaggccagc ggcggacggg tgaggaacac gtgggtaatc tgcctttcag 120 gccggaataa cgcccggaaa cgggcgctaa agccggatac gcccgcgagg aggcatcttc 180 ttgcggggga aggcccaatt gggtcgctga gagaggagcc cgcggcgcat tagctagttg 240 gcggggtaac ggcccaccaa ggcgacgatg cgtagccgac ctgagagggt gaccggccac 300 actgggactg agacacggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa 360 tgggcgcaag cctgacggag caacgccgcg tgagcgaaga aggccttcgg gttgtaaagc 420 tctgttgctc ggggagagcg gcatggggga tggaaagccc cgtgcgagac ggtaccgagt 480 gaggaagccc cggctaacta cgtgccagca gccgcggtaa aacgtagggg gcgagcgttg 540 tccggaatca ctgggcgtaa agggtgcgta ggcggtcgag caagtctgga gtgaaagtcc 600 atggctcaac catgggatgg ctttggaaac tgcttgactt gagtgctgga gaggcaaggg 660 gaattccacg tgtagcggtg aaatgcgtag agatgtggag gaataccagt ggcgaargcg 720 ccttgctgga cagtgactga cgctgaggca cgaaagcgtg gggagcaaac aggattagat 780 accctggtag tccacgccgt aaacgatgag tgctaggtgt tggggggaca caccccagtg 840 ccgaaggaaa mccaataagc actccgcctg gggagtacgg tcgcaagact gaaactcaaa 900 ggaattgacg ggggcccgca caagcagtgg agcatgtggt ttaaatcgaa gcaacgcgaa 960 gaaccttacc agggcttgac atccctctga caccctcaga gatgaggggt cccttcgggg 1020 cagaggagac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttcagt 1080 cccgcaacga gcgcaaccct tgacctgtgt taccagcgcg ttgaggcggg gactcacagg 1140 tgactgccgg cgtaagtcgg aggaaggcgg ggatgacgtc aaatcatcat gcccctgatg 1200 tcctgggcta cacacgtgct acaatgggcg gaacaaaggg aggcgaagcc gcgaggcgga 1260 gcgaaaccca aaaagccgct cgtagttcgg attgcaggct gcaactcgcc tgcatgaagc 1320 cggaattgct agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac 1380 acaccgcccg tcacaccacg agagtcggca acacccgaag tcggtgaggt aacccctgtg 1440 gggagccagc cgccgaaggt ggggtcgatg attggggtga agtcgtaaca aggtagccgt 1500 75 1519 DNA Alicyclobacillus acidoterrestris modified_base (549)..(549) a, c, g, or t 75 gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc gagcccttcg gggctagcgg 60 cggacgggtg agtaacacgt gggcaatccg cctttcagac tggaataaca ctcggaaacg 120 ggtgctaatg ccggataata cacgggtagg catctacttg tgttgaaaga tgcaactgca 180 tcgctgagag aggagcccgc ggcgcattag ctagttggtg aggtaacggc tcaccaaggc 240 gacgatgcgt agccgacctg agagggtgac cggccacact gggactgaga cacggcccag 300 actcctacgg gaggcagcag tagggaatct tccgcaatgg gcgcaagcct gacggagcaa 360 cgccgcgtga gcgaagaagg ccttcgggtt gtaaagctct gttgctcggg gagagcgaca 420 aggagagtgg aaagctcctt gtgagacggt accgagtgag gaagccccgg ctaactacgt 480 gccagcagcc gcggtaatac gtagggggca agcgttgtcc ggaatcactg gggcgtaaag 540 cgtgcgtang cggttgtgta agtctgaact gaaagtccaa ggctcnacct tgggnatgct 600 ttggaaactg catggacttg agtgctggag aggcnaggcn aattccncgt gttaccggtg 660 naaatgcgnt anatatgtgg aggaatacca gtggcnaang cgcctttgct ggacagtgga 720 ctgacgctga aggcacgaaa ancgtgggga ncaacnggat tanatccccn aangcgnggg 780 gaagcaaaca ggattagatt cccnttgtag tcccgccccg taancnatga gtacttagtt 840 gttgggggaa cacaccccan tgcggnggaa acccaataag cactccgcct ggggagtgcg 900 gtcncaagac tgaanctcaa aggaattgac gggggcccgc acaagcagtg gagcatntgg 960 tttaattcga agcaacgcga agaaccttac cagggctnga catccctctg accggtgcag 1020 agatgtacct tcccttcggg gcagaggaga caggtggtgc atggttgtcg tcagctcgtg 1080 tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttgatctgtg ttaccagcac 1140 gttgtggtgg ggactcacag gtgactgccg gcgtaagtcg gaggaaggcg gggatgacgt 1200 caaatcatca tgccctttat gtcctgggct acacacgtgc tacaatgggc ggtacaacgg 1260 gaagcgaagc cgcgaggtgg agcaaaacct aaaaagccgt tcgtagttcg gattgcaggc 1320 tgcaactcgc ctgcatgaag ccggaattgc tagtaatcgc ggatcagcat gccgcggtga 1380 atccgttccc gggccttgta cacaccgccc gtcacaccac gagagtcggc aacacccgaa 1440 gtcggtgagg taaccgttat ggagccagcc gccgaaggtg gggttgatga ttggggtgaa 1500 gtcgtaacaa ggtagccgt 1519 76 1497 DNA Alicyclobacillus cycloheptanicus modified_base (967) a, t, c or g 76 agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60 ggacccttcg gggtcagcgg cggacgggtg agtaacacgt gggtaatctg cccaactgac 120 cggaataacg cctggaaacg ggtgctaatg ccggataggc agcgagcagg catctgctcg 180 ctgggaaagg tgcaagtgca ccgcagatgg aggagcccgc ggcgcattag ctggttggtg 240 gggtaacggc tcaccaaggc gacgatgcgt agccgacctg agagggtgga cggccacact 300 gggactgaga cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg 360 gcgcaagcct gacggagcaa cgccgcgtga gcgaagaagg ccttcgggtt gtaaagctca 420 gtcactcggg aagagcggca aggggagtgg aaagcccctt gagagacggt accgagagag 480 gaagccccgg ctaactacgt gccagcagcc gcggtaatac gtagggggca agcgttgtcc 540 ggaatcactg ggcgtaaagc gtgcgtaggc ggttgcgtgt gtccggggtg aaagtccagg 600 gctcaaccct gggaatgcct tggaaactgc gtaacttgag tgctggagag gcaaggggaa 660 ttccgcgtgt agcggtggaa tgcgtagata tgcggaggaa taccagtggc gaaggcgcct 720 tgctggacag tgactgacgc tgaggcacga aagcgtgggg agcaaacagg attagatacc 780 ctggtagtcc acgccgtaaa cgatgagtgc taggtgttgg ggggtaccac cctcagtgcc 840 gaaggaaacc caataagcac tccgcctggg gagtacggtc gcaagactga aactcaaagg 900 aattgacggg ggcccgcaca agcagtggag catgtggttt aattcgaagc aacgcgaaga 960 accttancag ggctcgacat ccccctgaca gccgcagaga tgcggtttcc cttcggggca 1020 ggggagacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1080 cgcaacgagc gcaacccttg aactgtgtta ccagcacgtg aaggtgggga ctcacagttg 1140 actgccggcg taagtcggag gaaggcgggg atgacgtcaa atcatcatgc cctttatgtc 1200 ctgggctaca cacgtgctac aatgggcggt acaacgggaa gcgagaccgc gaggtggagc 1260 aaacccctga aagccgttcg tagttcggat tgcaggctgc aactcgcctg catgaagccg 1320 gaattgctag taatcgcgga tcagcatgcc gcggtgaatc cgttcccggg ccttgtacac 1380 accgcccgtc acaccacgag agtcggcaac acccgaagtc ggtggggtaa cccgtcaggg 1440 agccagccgc cgaaggtggg gttgatgatt ggggtgaagt cgtaacaagg tagccgt 1497 77 1546 DNA Artificial Sequence Description of Artificial Sequence Figure 1 consensus sequence 77 nnnnnnnnnn nnnnnnnnnn gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60 gnnncncttc ggnggnnagc ggcggacggg tgagnaacac gtgggnaatc ngccnnncng 120 ncnggaataa cncnnggaaa cgggngctaa ngccggatan nnnnncgngn aggcatctnc 180 tngnnnngna agnnncaant gnnncgcnga nngaggagcc cgcggcgcat tagctngttg 240 gngnggtaac ggcncaccaa ggcgacgatg cgtagccgac ctgagagggt gnncggccac 300 actgggactg agacacggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa 360 tgggcgcaag cctgacggag caacgccgcg tgagcgaaga aggccttcgg gttgtaaagc 420 tcngtnnctc gggnagagcg ncanggngnn tggaaagcnc cntgngagac ggtaccgagn 480 gaggaagccc cggctaacta cgtgccagca gccgcggtaa nacgtagggg gcnagcgttg 540 tccggaatca ctgggncgta aagngtgcgt angcggtngn gnnngtcngn nntgaaagtc 600 canggctcna ccntgggnnn gcnttggaaa ctgcntnnac ttgagtgctg gagaggcnag 660 gnnaattccn cgtgtnancg gtgnaantgc gnnananatg nggaggaata ccagtggcna 720 angcgccttn gctggacagt gnactgacgc tganggcacg aaanncgtgg ggancaannn 780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn acaggattag atncccnnng tagtccnncn 840 ccgtaancna tgagtnctna gntgttgggg gnnnncaccc ncantgcngn nggaaancca 900 ataagcactc cgcctgggga gtncggtcnc aagactgaan ctcaaaggaa ttgacggggg 960 cccgcacaag cagtggagca tntggtttaa ntcgaagcaa cgcgaagaac cttancaggg 1020 ctngacatcc cnctgacnnn nncagagatg nnnnntccct tcggggcagn ggagacaggt 1080 ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttnagtcccg caacgagcgc 1140 aacccttgan ctgtgttacc agcncgtnnn ggnggggact cacagntgac tgccggcgta 1200 agtcggagga aggcggggat gacgtcaaat catcatgccc ntnatgtcct gggctacaca 1260 cgtgctacaa tgggcggnac aangggangc gannccgcga ggnggagcna anccnnnaaa 1320 gccgntcgta gttcggattg caggctgcaa ctcgcctgca tgaagccgga attgctagta 1380 atcgcggatc agcatgccgc ggtgaatncg ttcccgggcc ttgtacacac cgcccgtcac 1440 accacgagag tcggcaacac ccgaagtcgg tgnggtaacc nntnnnngga gccagccgcc 1500 gaaggtgggg tngatgattg gggtgaagtc gtaacaaggt agccgt 1546 78 1498 DNA Artificial Sequence Description of Artificial Sequence Figure 1 majority sequence 78 agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60 gggcccttcg gggccagcgg cggacgggtg agtaacacgt gggtaatctg cctttcagac 120 cggaataacg cccggaaacg ggtgctaatg ccggatangc acgcgagnag gcatctnctt 180 gcggggaaag gtgcaantgc atcgctgaga gaggagcccg cggcgcatta gctagttggt 240 ggggtaacgg ctcaccaagg cgacgatgcg tagccgacct gagagggtga ccggccacac 300 tgggactgag acacggccca gactcctacg ggaggcagca gtagggaatc ttccgcaatg 360 ggcgcaagcc tgacggagca acgccgcgtg agcgaagaag gccttcgggt tgtaaagctc 420 tgttgctcgg ggagagcggc aaggggagtg gaaagcccct tgngagacgg taccgagtga 480 ggaagccccg gctaactacg tgccagcagc cgcggtaata cgtagggggc aagcgttgtc 540 cggaatcact gggcgtaaag cgtgcgtagg cggttgngta agtctggagt gaaagtccan 600 ggctcaaccn tgggaatgct ttggaaactg cntgacttga gtgctggaga ggcaagggga 660 attccncgtg tagcggtgna atgcgtagat atgtggagga ataccagtgg cgaangcgcc 720 ttgctggaca gtgactgacg ctgaggcacg aaagcgtggg gagcaaacag gattagatac 780 cctggtagtc cacgccgtaa acgatgagtg ctaggtgttg gggggacaca ccccagtgcc 840 gaaggaaacc caataagcac tccgcctggg gagtacggtc gcaagactga aactcaaagg 900 aattgacggg ggcccgcaca agcagtggag catgtggttt aattcgaagc aacgcgaaga 960 accttaccag ggctngacat ccctctgaca gccgcagaga tgnggnttcc cttcggggca 1020 gaggagacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1080 cgcaacgagc gcaacccttg anctgtgtta ccagcacgtt gaggtgggga ctcacaggtg 1140 actgccggcg taagtcggag gaaggcgggg atgacgtcaa atcatcatgc cctttatgtc 1200 ctgggctaca cacgtgctac aatgggcggt acaacgggaa gcgaagccgc gaggtggagc 1260 aaaacccaaa aagccgttcg tagttcggat tgcaggctgc aactcgcctg catgaagccg 1320 gaattgctag taatcgcgga tcagcatgcc gcggtgaatc cgttcccggg ccttgtacac 1380 accgcccgtc acaccacgag agtcggcaac acccgaagtc ggtgaggtaa cccntntngg 1440 gagccagccg ccgaaggtgg ggttgatgat tggggtgaag tcgtaacaag gtagccgt 1498 79 718 DNA Alicyclobacillus acidocaldarius 79 gggggttgga tgttacaggc ttccatctcg cccgtgtggg acacgggtct cgccgtgctc 60 gcgctgcgcg ctgcggggct tccggccgat cactgaccgg ttggtcaagg ctgggctgaa 120 tggctgttgg accggcagat caccgtgccg ggcgattggg tggtgaagcg cccgaacctc 180 aacccgggcg gcttcgcgct ccagttcgac aacgtgtact atccggacgt ggacgacacg 240 gccgtcgtca tctgggcgct caacacgctg cgactcccgg acgagcgccg caggcgagac 300 gccatgacga agggattccg gccatgacga agggattccg ctggattgtc ggcatgcaga 360 gctcgaacgg cggctggggc gcatacgacg tcgacaacac gagcgatctc ccgaaccaca 420 tcccgttctg cgacttcggc gaagtgaccg atccgccgtc ggaagacgtc accgcccacg 480 tgctcgagtg tttcggcagc ttcggctacg acgacgcctg gaaggtgatc cagcgcgcgg 540 tggcgtacct caagcgggag cagaagccgg acggcagctg gttcggtcgc tggggcgtca 600 actacgtgta tggcatcggc gcggtggtgt cggcgctgaa ggcggtcggg atcgacatgc 660 gcgagccgta cattcaaaag gcgctcgatt gggtggagca gcatcagaac ccggacgg 718 80 878 DNA Alicyclobacillus acidocaldarius 80 ggaggatgga tgtttcaggc ttccatctcg ccggtgtggg acacgggcct cgccgtgctc 60 gcgctgcgcg ctgcggggct tccggccgat cacgaccgct tggtcaaggc gggcgagtgg 120 ctgttggacc ggcagatcac ggttccgggc gactgggcgg tgaagcgccc gaacctcaag 180 ccgggcgggt tcgcgttcca gttcgacaac gtgtactacc cggacgtgga cgacacggcc 240 gtcgtggtgt gggcgctcaa caccctgcgc ttgccggacg agcgccgcag gcgggacgcc 300 atgacgaagg gattccgctg gattgtcggc atgcagagct cgaacggcgg ttggggcgcc 360 tacgacgtcg acaacacgag cgatctcccg aaccacatcc cgttctgcga cttcggcgaa 420 gtgaccgatc cgccgtcaga ggacgtcacc gcccacgtgc tcgagtgttt cggcagcttc 480 gggtacgatg acgcctggaa ggtcatccgg cgcgcggtgg aatatctcaa gcgggagcag 540 aagccggacg gcagctggtt cggtcgttgg ggcgtcaatt acctctacgg cacgggcgcg 600 gtggtgtcgg cgctgaaggc ggtcgggatc gacacgcgcg agccgtacat tcaaaaggcg 660 ctcgactggg tcgagcagca tcagaacccg gacggcggct ggggcgagga ctgccgctcg 720 tacgaggatc cggcgtacgc gggtaagggc gcgagcaccc cgtcgcagac ggcctgggcg 780 ctgatggcgc tcatcgcggg cggcagggcg gagtccgagg ccgcgcgccg cggcgtgcaa 840 tacctcgtgg agacgcagcg cccggacggc ggctggga 878 81 878 DNA Alicyclobacillus acidoterrestris 81 gggggttgga tgttccaggc gagtatttct ccaatctggg atactggctt gaccgtcttg 60 gcactgcgtt cggctggatt gccaccagat catccagcgc tgattaaagc gggtgagtgg 120 ttggtcagta aacaaattct caaggatggc gactggaaag ttcgtcgacg caaggcgaaa 180 ccaggcggtt gggcatttga attccactgc gaaaactacc cagacgtcga cgatacggcg 240 atggtcgtct tggcgctcaa tggcattcaa ttgccggatg aagggaagcg tcgtgacgca 300 ttgacccgtg gcttccgttg gttgcgcgag atgcagagtt cgaacggggg ctggggcgca 360 tacgatgtgg acaacacgcg tcagttgacc aatcggattc cattttgcaa cttcggcgaa 420 gtgattgatc cgccatcgga agacgtcacc gcacacgtct tggagtgctt cggcagcttt 480 gggtacgacg aggcatggaa ggtgattcgc aaggcggtcg agtatctcaa ggcgcaacaa 540 cgcccagatg ggtcatggtt tggccgctgg ggcgtcaact acgtgtatgg catcggcgcg 600 gtcgttccgg gactcaaggc cgtcggtgtc gatatgcgtg agccgtgggt gcaaaagtcg 660 ctcgactggc tcgtcgagca tcaaaatgag gatggcggct ggggtgaaag ccgaattcca 720 gcacactggc ggccgttact agtggatccg agctcggtac caagcttggc gtaatcatgg 780 tcatagctgt ttcctgtgtg aaattggtat ccgctcacaa ttcacacaac atacgagccg 840 gaacataagt gtaagcctgg ggtgcctatg agtgagct 878 82 878 DNA Alicyclobacillus acidocaldarius 82 gggggttgga tgttccaggc gagtatttct ccaatctggg atactggctt gaccgtcttg 60 gcactgcgtt cggctggatt gccaccagat catccagcgc tgattaaagc gggtgagtgg 120 ttggtcagta aacaaattct caaggatggc gactggaaag ttcgtcgacg caaggcgaaa 180 ccaggcggtt gggcatttga attccactgc gaaaactacc cagacgtcga cgatacggcg 240 atggtcgtct tggcgctcaa tggcattcaa ttgccggatg aagggaagcg tcgtgacgca 300 ttgacccgtg gcttccgttg gttgcgcgag atgcagagtt cgaacggggg ctggggcgca 360 tacgatgtgg acaacacgcg tcagttgacc aaatcggatt ccatttttgc gacttcgggc 420 gaagtgattg atccgccatc ggaagacgtc accgcacacg tcttggagtg cttcggcagc 480 tttgggtacg acgaggcatg gaaggtgatt cgcaaggcgg tcgagtatct caaggcgcaa 540 caacgcccag atgggtcatg gtttggccgc tggggcgtca actacgtgta tggcatcggc 600 gcggtcgttc cgggactcaa ggccgtcggt gtcgatatgc gtgagccgtg ggtgcaaaag 660 tcgctcgact ggctcgtcga gcatcaaaat gaggatggcg gttggggtga agattgccgt 720 tcctatgatg atccacgtct cgcaggtcag ggtgtgagta caccgtcgca gaccgcctgg 780 gcgttgatgg cgctcatcgc gggcggccgt gtcgagtcag atgcggtatt gcgcggggtc 840 acttaccttc acgacacgca gcgcgcagat ggtggctg 878 83 631 PRT Alicyclobacillus acidocaldarius 83 Met Ala Glu Gln Leu Val Glu Ala Pro Ala Tyr Ala Arg Thr Leu Asp 1 5 10 15 Arg Ala Val Glu Tyr Leu Leu Ser Cys Gln Lys Asp Glu Gly Tyr Trp 20 25 30 Trp Gly Pro Leu Leu Ser Asn Val Thr Met Glu Ala Glu Tyr Val Leu 35 40 45 Leu Cys His Ile Leu Asp Arg Val Asp Arg Asp Arg Met Glu Lys Ile 50 55 60 Arg Arg Tyr Leu Leu His Glu Gln Arg Glu Asp Gly Thr Trp Ala Leu 65 70 75 80 Tyr Pro Gly Gly Pro Pro Asp Leu Asp Thr Thr Ile Glu Ala Tyr Val 85 90 95 Ala Leu Lys Tyr Ile Gly Met Ser Arg Asp Glu Glu Pro Met Gln Lys 100 105 110 Ala Leu Arg Phe Ile Gln Ser Gln Gly Gly Ile Glu Ser Ser Arg Val 115 120 125 Phe Thr Arg Met Trp Leu Ala Leu Val Gly Glu Tyr Pro Trp Glu Lys 130 135 140 Val Pro Met Val Pro Pro Glu Ile Met Phe Leu Gly Lys Arg Met Pro 145 150 155 160 Leu Asn Ile Tyr Glu Phe Gly Ser Trp Ala Arg Ala Thr Val Val Ala 165 170 175 Leu Ser Ile Val Met Ser Arg Gln Pro Val Phe Pro Leu Pro Glu Arg 180 185 190 Ala Arg Val Pro Glu Leu Tyr Glu Thr Asp Val Pro Pro Arg Arg Arg 195 200 205 Gly Ala Lys Gly Gly Gly Gly Trp Ile Phe Asp Ala Leu Asp Arg Ala 210 215 220 Leu His Gly Tyr Gln Lys Leu Ser Val His Pro Phe Arg Arg Ala Ala 225 230 235 240 Glu Ile Arg Ala Leu Asp Trp Leu Leu Glu Arg Gln Ala Gly Asp Gly 245 250 255 Ser Trp Gly Gly Ile Gln Pro Pro Trp Phe Tyr Ala Leu Ile Ala Leu 260 265 270 Lys Ile Leu Asp Met Thr Gln His Pro Ala Phe Ile Lys Gly Trp Glu 275 280 285 Gly Leu Glu Leu Tyr Gly Val Glu Leu Asp Tyr Gly Gly Trp Met Phe 290 295 300 Gln Ala Ser Ile Ser Pro Val Trp Asp Thr Gly Leu Ala Val Leu Ala 305 310 315 320 Leu Arg Ala Ala Gly Leu Pro Ala Asp His Asp Arg Leu Val Lys Ala 325 330 335 Gly Glu Trp Leu Leu Asp Arg Gln Ile Thr Val Pro Gly Asp Trp Ala 340 345 350 Val Lys Arg Pro Asn Leu Lys Pro Gly Gly Phe Ala Phe Gln Phe Asp 355 360 365 Asn Val Tyr Tyr Pro Asp Val Asp Asp Thr Ala Val Val Val Trp Ala 370 375 380 Leu Asn Thr Leu Arg Leu Pro Asp Glu Arg Arg Arg Arg Asp Ala Met 385 390 395 400 Thr Lys Gly Phe Arg Trp Ile Val Gly Met Gln Ser Ser Asn Gly Gly 405 410 415 Trp Gly Ala Tyr Asp Val Asp Asn Thr Ser Asp Leu Pro Asn His Ile 420 425 430 Pro Phe Cys Asp Phe Gly Glu Val Thr Asp Pro Pro Ser Glu Asp Val 435 440 445 Thr Ala His Val Leu Glu Cys Phe Gly Ser Phe Gly Tyr Asp Asp Ala 450 455 460 Trp Lys Val Ile Arg Arg Ala Val Glu Tyr Leu Lys Arg Glu Gln Lys 465 470 475 480 Pro Asp Gly Ser Trp Phe Gly Arg Trp Gly Val Asn Tyr Leu Tyr Gly 485 490 495 Thr Gly Ala Val Val Ser Ala Leu Lys Ala Val Gly Ile Asp Thr Arg 500 505 510 Glu Pro Tyr Ile Gln Lys Ala Leu Asp Trp Val Glu Gln His Gln Asn 515 520 525 Pro Asp Gly Gly Trp Gly Glu Asp Cys Arg Ser Tyr Glu Asp Pro Ala 530 535 540 Tyr Ala Gly Lys Gly Ala Ser Thr Pro Ser Gln Thr Ala Trp Ala Leu 545 550 555 560 Met Ala Leu Ile Ala Gly Gly Arg Ala Glu Ser Glu Ala Ala Arg Arg 565 570 575 Gly Val Gln Tyr Leu Val Glu Thr Gln Arg Pro Asp Gly Gly Trp Asp 580 585 590 Glu Pro Tyr Tyr Thr Gly Thr Ala Ser Pro Gly Asp Phe Tyr Leu Gly 595 600 605 Tyr Thr Met Tyr Arg His Val Phe Pro Thr Leu Ala Leu Gly Arg Tyr 610 615 620 Lys Gln Ala Ile Glu Arg Arg 625 630 84 631 PRT Alicyclobacillus acidocaldarius 84 Met Ala Glu Gln Leu Val Glu Ala Pro Ala Tyr Ala Arg Thr Leu Asp 1 5 10 15 Arg Ala Val Glu Tyr Leu Leu Ser Cys Gln Lys Asp Glu Gly Tyr Trp 20 25 30 Trp Gly Pro Leu Leu Ser Asn Val Thr Met Glu Ala Glu Tyr Val Leu 35 40 45 Leu Cys His Ile Leu Asp Arg Val Asp Arg Asp Arg Met Glu Lys Ile 50 55 60 Arg Arg Tyr Leu Leu His Glu Gln Arg Glu Asp Gly Thr Trp Ala Leu 65 70 75 80 Tyr Pro Gly Gly Pro Pro Asp Leu Asp Thr Thr Ile Glu Ala Tyr Val 85 90 95 Ala Leu Lys Tyr Ile Gly Met Ser Arg Asp Glu Glu Pro Met Gln Lys 100 105 110 Ala Leu Arg Phe Ile Gln Ser Gln Gly Gly Ile Glu Ser Ser Arg Val 115 120 125 Phe Thr Arg Met Trp Leu Ala Leu Val Gly Glu Tyr Pro Trp Glu Lys 130 135 140 Val Pro Met Val Pro Pro Glu Ile Met Phe Leu Gly Lys Arg Met Pro 145 150 155 160 Leu Asn Ile Tyr Glu Phe Gly Ser Trp Ala Arg Ala Thr Val Val Ala 165 170 175 Leu Ser Ile Val Met Ser Arg Gln Pro Val Phe Pro Leu Pro Glu Arg 180 185 190 Ala Arg Val Pro Glu Leu Tyr Glu Thr Asp Val Pro Pro Arg Arg Arg 195 200 205 Gly Ala Lys Gly Gly Gly Gly Trp Ile Phe Asp Ala Leu Asp Arg Ala 210 215 220 Leu His Gly Tyr Gln Lys Leu Ser Val His Pro Phe Arg Arg Ala Ala 225 230 235 240 Glu Ile Arg Ala Leu Asp Trp Leu Leu Glu Arg Gln Ala Gly Asp Gly 245 250 255 Ser Trp Gly Gly Ile Gln Pro Pro Trp Phe Tyr Ala Leu Ile Ala Leu 260 265 270 Lys Ile Leu Asp Met Thr Gln His Pro Ala Phe Ile Lys Gly Trp Glu 275 280 285 Gly Leu Glu Leu Tyr Gly Val Glu Leu Asp Tyr Gly Gly Trp Met Phe 290 295 300 Gln Ala Ser Ile Ser Pro Val Trp Asp Thr Gly Leu Ala Val Leu Ala 305 310 315 320 Leu Arg Ala Ala Gly Leu Pro Ala Asp His Asp Arg Leu Val Lys Ala 325 330 335 Gly Glu Trp Leu Leu Asp Arg Gln Ile Thr Val Pro Gly Asp Trp Ala 340 345 350 Val Lys Arg Pro Asn Leu Lys Pro Gly Gly Phe Ala Phe Gln Phe Asp 355 360 365 Asn Val Tyr Tyr Pro Asp Val Asp Asp Thr Ala Val Val Val Trp Ala 370 375 380 Leu Asn Thr Leu Arg Leu Pro Asp Glu Arg Arg Arg Arg Asp Ala Met 385 390 395 400 Thr Lys Gly Phe Arg Trp Ile Val Gly Met Gln Ser Ser Asn Gly Gly 405 410 415 Trp Gly Ala Tyr Asp Val Asp Asn Thr Ser Asp Leu Pro Asn His Ile 420 425 430 Pro Phe Cys Asp Phe Gly Glu Val Thr Asp Pro Pro Ser Glu Asp Val 435 440 445 Thr Ala His Val Leu Glu Cys Phe Gly Ser Phe Gly Tyr Asp Asp Ala 450 455 460 Trp Lys Val Ile Arg Arg Ala Val Glu Tyr Leu Lys Arg Glu Gln Lys 465 470 475 480 Pro Asp Gly Ser Trp Phe Gly Arg Trp Gly Val Asn Tyr Leu Tyr Gly 485 490 495 Thr Gly Ala Val Val Ser Ala Leu Lys Ala Val Gly Ile Asp Thr Arg 500 505 510 Glu Pro Tyr Ile Gln Lys Ala Leu Asp Trp Val Glu Gln His Gln Asn 515 520 525 Pro Asp Gly Gly Trp Gly Glu Asp Cys Arg Ser Tyr Glu Asp Pro Ala 530 535 540 Tyr Ala Gly Lys Gly Ala Ser Thr Pro Ser Gln Thr Ala Trp Ala Leu 545 550 555 560 Met Ala Leu Ile Ala Gly Gly Arg Ala Glu Ser Glu Ala Ala Arg Arg 565 570 575 Gly Val Gln Tyr Leu Val Glu Thr Gln Arg Pro Asp Gly Gly Trp Asp 580 585 590 Glu Pro Tyr Tyr Thr Gly Thr Gly Phe Pro Gly Asp Phe Tyr Leu Gly 595 600 605 Tyr Thr Met Tyr Arg His Val Phe Pro Thr Leu Ala Leu Gly Arg Tyr 610 615 620 Lys Gln Ala Ile Glu Arg Arg 625 630 85 634 PRT Alicyclobacillus acidoterrestris 85 Met Thr Lys Gln Leu Leu Asp Thr Pro Met Val Gln Ala Thr Leu Glu 1 5 10 15 Ala Gly Val Ala His Leu Leu Arg Arg Gln Ala Pro Asp Gly Tyr Trp 20 25 30 Trp Ala Pro Leu Leu Ser Asn Val Cys Met Glu Ala Glu Tyr Val Leu 35 40 45 Leu Cys His Cys Leu Gly Lys Lys Asn Pro Glu Arg Glu Ala Gln Ile 50 55 60 Arg Lys Tyr Ile Ile Ser Gln Arg Arg Glu Asp Gly Thr Trp Ser Ile 65 70 75 80 Tyr Pro Gly Gly Pro Ser Asp Leu Asn Ala Thr Val Glu Ala Tyr Val 85 90 95 Ala Leu Lys Tyr Leu Gly Glu Pro Ala Ser Asp Pro Gln Met Val Gln 100 105 110 Ala Lys Glu Phe Ile Gln Asn Glu Gly Gly Ile Glu Ser Thr Arg Val 115 120 125 Phe Thr Arg Leu Trp Leu Ala Met Val Gly Gln Tyr Pro Trp Asp Lys 130 135 140 Leu Pro Val Ile Pro Pro Glu Ile Met His Leu Pro Lys Ser Val Pro 145 150 155 160 Leu Asn Ile Tyr Asp Phe Ala Ser Trp Ala Arg Ala Thr Ile Val Thr 165 170 175 Leu Ser Tyr Arg His Glu Ser Pro Thr Cys Asp Ala Thr Ser Gly Leu 180 185 190 Cys Lys Gly Ser Gly Ile Val Arg Gly Glu Gly Pro Pro Lys Arg Arg 195 200 205 Ser Ala Lys Gly Gly Asp Ser Gly Phe Phe Val Ala Leu Asp Lys Phe 210 215 220 Leu Lys Ala Tyr Asn Lys Trp Pro Ile Gln Pro Gly Arg Lys Ser Gly 225 230 235 240 Glu Gln Lys Ala Leu Glu Trp Ile Leu Ala His Gln Glu Ala Asp Gly 245 250 255 Cys Trp Gly Gly Ile Gln Pro Pro Trp Phe Tyr Ala Leu Leu Ala Leu 260 265 270 Lys Cys Leu Asn Met Thr Asp His Pro Ala Phe Val Lys Gly Phe Glu 275 280 285 Gly Leu Glu Ala Tyr Gly Val His Thr Ser Asp Gly Gly Trp Met Phe 290 295 300 Gln Ala Ser Ile Ser Pro Ile Trp Asp Thr Gly Leu Thr Val Leu Ala 305 310 315 320 Leu Arg Ser Ala Gly Leu Pro Pro Asp His Pro Ala Leu Ile Lys Ala 325 330 335 Gly Glu Trp Leu Val Ser Lys Gln Ile Leu Lys Asp Gly Asp Trp Lys 340 345 350 Val Arg Arg Arg Lys Ala Lys Pro Gly Gly Trp Ala Phe Glu Phe His 355 360 365 Cys Glu Asn Tyr Pro Asp Val Asp Asp Thr Ala Met Val Val Leu Ala 370 375 380 Leu Asn Gly Ile Gln Leu Pro Asp Glu Gly Lys Arg Arg Asp Ala Leu 385 390 395 400 Thr Arg Gly Phe Arg Trp Leu Arg Glu Met Gln Ser Ser Asn Gly Gly 405 410 415 Trp Gly Ala Tyr Asp Val Asp Asn Thr Arg Gln Leu Thr Lys Ser Asp 420 425 430 Ser Ile Phe Ala Thr Ser Gly Glu Val Ile Asp Pro Pro Ser Glu Asp 435 440 445 Val Thr Ala His Val Leu Glu Cys Phe Gly Ser Phe Gly Tyr Asp Glu 450 455 460 Ala Trp Lys Val Ile Arg Lys Ala Val Glu Tyr Leu Lys Ala Gln Gln 465 470 475 480 Arg Pro Asp Gly Ser Trp Phe Gly Arg Trp Gly Val Asn Tyr Val Tyr 485 490 495 Gly Ile Gly Ala Val Val Pro Gly Leu Lys Ala Val Gly Val Asp Met 500 505 510 Arg Glu Pro Trp Val Gln Lys Ser Leu Asp Trp Leu Val Glu His Gln 515 520 525 Asn Glu Asp Gly Gly Trp Gly Glu Asp Cys Arg Ser Tyr Asp Asp Pro 530 535 540 Arg Leu Ala Gly Gln Gly Val Ser Thr Pro Ser Gln Thr Ala Trp Ala 545 550 555 560 Leu Met Ala Leu Ile Ala Gly Gly Arg Val Glu Ser Asp Ala Val Leu 565 570 575 Arg Gly Val Thr Tyr Leu His Asp Thr Gln Arg Ala Asp Gly Gly Trp 580 585 590 Asp Glu Glu Val Tyr Thr Gly Thr Gly Phe Pro Gly Asp Phe Tyr Leu 595 600 605 Ala Tyr Thr Met Tyr Arg Asp Ile Leu Pro Val Trp Ala Leu Gly Arg 610 615 620 Tyr Gln Glu Ala Met Gln Arg Ile Arg Gly 625 630 86 556 PRT Bacillus subtilis 86 Met Gly Thr Leu Gln Glu Lys Val Arg Arg Phe Gln Lys Lys Thr Ile 1 5 10 15 Thr Glu Leu Arg Asp Arg Gln Asn Ala Asp Gly Ser Trp Thr Phe Cys 20 25 30 Phe Glu Gly Pro Ile Met Thr Asn Ser Phe Phe Ile Leu Leu Leu Thr 35 40 45 Ser Leu Asp Glu Gly Glu Asn Glu Lys Glu Leu Ile Ser Ser Leu Ala 50 55 60 Ala Gly Ile His Ala Lys Gln Gln Pro Asp Gly Thr Phe Ile Asn Tyr 65 70 75 80 Pro Asp Glu Thr Arg Gly Asn Leu Thr Ala Thr Val Gln Gly Tyr Val 85 90 95 Gly Met Leu Ala Ser Gly Cys Phe His Arg Thr Glu Pro His Met Lys 100 105 110 Lys Ala Glu Gln Phe Ile Ile Ser His Gly Gly Leu Arg His Val His 115 120 125 Phe Met Thr Lys Trp Met Leu Ala Ala Asn Gly Leu Tyr Pro Trp Pro 130 135 140 Ala Leu Tyr Leu Pro Leu Ser Leu Met Ala Leu Pro Pro Thr Leu Pro 145 150 155 160 Ile His Phe Tyr Gln Phe Ser Ser Tyr Ala Arg Ile His Phe Ala Pro 165 170 175 Met Ala Val Thr Leu Asn Gln Arg Phe Val Leu Ile Asn Arg Asn Ile 180 185 190 Ser Ser Leu His His Leu Asp Pro His Met Thr Lys Asn Pro Phe Thr 195 200 205 Trp Leu Arg Ser Asp Ala Phe Glu Glu Arg Asp Leu Thr Ser Ile Leu 210 215 220 Leu His Trp Lys Arg Val Phe His Ala Pro Phe Ala Phe Gln Gln Leu 225 230 235 240 Gly Leu Gln Thr Ala Lys Thr Tyr Met Leu Asp Arg Ile Glu Lys Asp 245 250 255 Gly Thr Leu Tyr Ser Tyr Ala Ser Ala Thr Ile Tyr Met Val Tyr Ser 260 265 270 Leu Leu Ser Leu Gly Val Ser Arg Tyr Ser Pro Ile Ile Arg Arg Ala 275 280 285 Ile Thr Gly Ile Lys Ser Leu Val Thr Lys Cys Asn Gly Ile Pro Tyr 290 295 300 Leu Glu Asn Ser Thr Ser Thr Val Trp Asp Thr Ala Leu Ile Ser Tyr 305 310 315 320 Ala Leu Gln Lys Asn Gly Val Thr Glu Thr Asp Gly Ser Val Thr Lys 325 330 335 Ala Ala Asp Phe Leu Leu Glu Arg Gln His Thr Lys Ile Ala Asp Trp 340 345 350 Ser Val Lys Asn Pro Asn Ser Val Pro Gly Gly Trp Gly Phe Ser Asn 355 360 365 Ile Asn Thr Asn Asn Pro Asp Cys Asp Asp Thr Thr Ala Val Leu Lys 370 375 380 Ala Ile Pro Arg Asn His Ser Pro Ala Ala Trp Glu Arg Gly Val Ser 385 390 395 400 Trp Leu Leu Ser Met Gln Asn Asn Asp Gly Gly Phe Ser Ala Phe Glu 405 410 415 Lys Asn Val Asn His Pro Leu Ile Arg Leu Leu Pro Leu Glu Ser Ala 420 425 430 Glu Asp Ala Ala Val Asp Pro Ser Thr Ala Asp Leu Thr Gly Arg Val 435 440 445 Leu His Phe Leu Gly Glu Lys Val Gly Phe Thr Glu Lys His Gln His 450 455 460 Ile Gln Arg Ala Val Lys Trp Leu Phe Glu His Gln Glu Gln Asn Gly 465 470 475 480 Ser Trp Tyr Gly Arg Trp Gly Val Cys Tyr Ile Tyr Gly Thr Trp Ala 485 490 495 Ala Leu Thr Gly Met His Ala Cys Gly Leu Thr Glu Ser Ile Pro Val 500 505 510 Tyr Lys Arg Leu Cys Val Gly Ser Asn Pro Tyr Lys Met Met Thr Glu 515 520 525 Ala Gly Glu Asn Pro Ala Lys Ala Pro Lys Ser Lys His Met Tyr Arg 530 535 540 Phe Ile Glu Glu Pro Leu Tyr Lys Arg Pro Gly Leu 545 550 555 87 706 PRT Dictyostelium discoideum 87 Phe Thr Arg Met Thr Thr Thr Asn Trp Ser Leu Lys Val Asp Arg Gly 1 5 10 15 Arg Gln Thr Trp Glu Tyr Ser Gln Glu Lys Lys Glu Ala Thr Asp Val 20 25 30 Asp Ile His Leu Leu Arg Leu Lys Glu Pro Gly Thr His Cys Pro Glu 35 40 45 Gly Cys Asp Leu Asn Arg Ala Lys Thr Pro Gln Gln Ala Ile Lys Lys 50 55 60 Ala Phe Gln Tyr Phe Ser Lys Val Gln Thr Glu Asp Gly His Trp Ala 65 70 75 80 Gly Asp Tyr Gly Gly Pro Met Phe Leu Leu Pro Gly Leu Val Ile Thr 85 90 95 Cys Tyr Val Thr Gly Tyr Gln Leu Pro Glu Ser Thr Gln Arg Glu Ile 100 105 110 Ile Arg Tyr Leu Phe Asn Arg Gln Asn Pro Val Asp Gly Gly Trp Gly 115 120 125 Leu His Ile Glu Ala His Ser Asp Ile Phe Gly Thr Thr Leu Gln Tyr 130 135 140 Val Ser Leu Arg Leu Leu Gly Val Pro Ala Asp His Pro Ser Val Val 145 150 155 160 Lys Ala Arg Thr Phe Leu Leu Gln Asn Gly Gly Ala Thr Gly Ile Pro 165 170 175 Ser Trp Gly Lys Phe Trp Leu Ala Thr Leu Asn Ala Tyr Asp Trp Asn 180 185 190 Gly Leu Asn Pro Ile Pro Ile Glu Phe Trp Leu Leu Pro Tyr Asn Leu 195 200 205 Pro Ile Ala Pro Gly Arg Trp Trp Cys His Cys Arg Met Val Tyr Leu 210 215 220 Pro Met Ser Tyr Ile Tyr Ala Lys Lys Thr Thr Gly Pro Leu Thr Asp 225 230 235 240 Leu Val Lys Asp Leu Arg Arg Glu Ile Tyr Cys Gln Glu Tyr Glu Lys 245 250 255 Ile Asn Trp Ser Glu Gln Arg Asn Asn Ile Ser Lys Leu Asp Met Tyr 260 265 270 Tyr Glu His Thr Ser Leu Leu Asn Val Ile Asn Gly Ser Leu Asn Ala 275 280 285 Tyr Glu Lys Val His Ser Lys Trp Leu Arg Asp Lys Ala Ile Asp Tyr 290 295 300 Thr Phe Asp His Ile Arg Tyr Glu Asp Glu Gln Thr Lys Tyr Ile Asp 305 310 315 320 Ile Gly Pro Val Asn Lys Thr Val Asn Met Leu Cys Val Trp Asp Arg 325 330 335 Glu Gly Lys Ser Pro Ala Phe Tyr Lys His Ala Asp Arg Leu Lys Asp 340 345 350 Tyr Leu Trp Leu Ser Phe Asp Gly Met Lys Met Gln Gly Tyr Asn Gly 355 360 365 Ser Gln Leu Trp Asp Thr Ala Phe Thr Ile Gln Ala Phe Met Glu Ser 370 375 380 Gly Ile Ala Asn Gln Phe Gln Asp Cys Met Lys Leu Ala Gly His Tyr 385 390 395 400 Leu Asp Ile Ser Gln Val Pro Glu Asp Ala Arg Asp Met Lys His Tyr 405 410 415 His Arg His Tyr Ser Lys Gly Ala Trp Pro Phe Ser Thr Val Asp His 420 425 430 Gly Trp Pro Ile Ser Asp Cys Thr Ala Glu Gly Ile Lys Ser Ala Leu 435 440 445 Ala Leu Arg Ser Leu Pro Phe Ile Glu Pro Ile Ser Leu Asp Arg Ile 450 455 460 Ala Asp Gly Ile Asn Val Leu Leu Thr Leu Gln Asn Gly Asp Gly Gly 465 470 475 480 Trp Ala Ser Tyr Glu Asn Thr Arg Gly Pro Lys Trp Leu Glu Lys Phe 485 490 495 Asn Pro Ser Glu Val Phe Gln Asn Ile Met Ile Asp Tyr Ser Tyr Val 500 505 510 Glu Cys Ser Ala Ala Cys Ile Gln Ala Met Ser Ala Phe Arg Lys His 515 520 525 Ala Pro Asn His Pro Arg Ile Lys Glu Ile Asn Arg Ser Ile Ala Arg 530 535 540 Gly Val Lys Phe Ile Lys Ser Ile Gln Arg Gln Asp Gly Ser Trp Leu 545 550 555 560 Gly Ser Trp Gly Ile Cys Phe Thr Tyr Gly Thr Trp Phe Gly Ile Glu 565 570 575 Gly Leu Val Ala Ser Gly Glu Pro Leu Thr Ser Pro Ser Ile Val Lys 580 585 590 Ala Cys Lys Phe Leu Ala Ser Lys Gln Arg Ala Asp Gly Gly Trp Gly 595 600 605 Glu Ser Phe Lys Ser Asn Val Thr Lys Glu Tyr Val Gln His Glu Thr 610 615 620 Ser Gln Val Val Asn Thr Gly Trp Ala Leu Leu Ser Leu Met Ser Ala 625 630 635 640 Lys Tyr Pro Asp Arg Glu Cys Ile Glu Arg Gly Ile Lys Phe Leu Ile 645 650 655 Gln Arg Gln Tyr Pro Asn Gly Asp Phe Pro Gln Glu Ser Ile Ile Gly 660 665 670 Val Phe Asn Phe Asn Cys Met Ile Ser Tyr Ser Asn Tyr Lys Asn Ile 675 680 685 Phe Pro Leu Trp Ala Leu Ser Arg Tyr Asn Gln Leu Tyr Leu Lys Ser 690 695 700 Lys Ile 705 88 647 PRT Synechocystis PCC6803 88 Met Val Ile Ala Ala Ser Pro Ser Val Pro Cys Pro Ser Thr Glu Gln 1 5 10 15 Val Arg Gln Ala Ile Ala Ala Ser Arg Asp Phe Leu Leu Ser Glu Gln 20 25 30 Tyr Ala Asp Gly Tyr Trp Trp Ser Glu Leu Glu Ser Asn Val Thr Ile 35 40 45 Thr Ala Glu Val Val Ile Leu His Lys Ile Trp Gly Thr Ala Ala Gln 50 55 60 Arg Pro Leu Glu Lys Ala Lys Asn Tyr Leu Leu Gln Gln Gln Arg Asp 65 70 75 80 His Gly Gly Trp Glu Leu Tyr Tyr Gly Asp Gly Gly Glu Leu Ser Thr 85 90 95 Ser Val Glu Ala Tyr Thr Ala Leu Arg Ile Leu Gly Val Pro Ala Thr 100 105 110 Asp Pro Ala Leu Val Lys Ala Lys Asn Phe Ile Val Gly Arg Gly Gly 115 120 125 Ile Ser Lys Ser Arg Ile Phe Thr Lys Met His Leu Ala Leu Ile Gly 130 135 140 Cys Tyr Asp Trp Arg Gly Thr Pro Ser Ile Pro Pro Trp Val Met Leu 145 150 155 160 Leu Pro Asn Asn Phe Phe Phe Asn Ile Tyr Glu Met Ser Ser Trp Ala 165 170 175 Arg Ser Ser Thr Val Pro Leu Met Ile Val Cys Asp Gln Lys Pro Val 180 185 190 Tyr Asp Ile Ala Gln Gly Leu Arg Val Asp Glu Leu Tyr Ala Glu Gly 195 200 205 Met Glu Asn Val Gln Tyr Lys Leu Pro Glu Ser Gly Thr Ile Trp Asp 210 215 220 Ile Phe Ile Gly Leu Asp Ser Leu Phe Lys Leu Gln Glu Gln Ala Lys 225 230 235 240 Val Val Pro Phe Arg Glu Gln Gly Leu Ala Leu Ala Glu Lys Trp Ile 245 250 255 Leu Glu Arg Gln Glu Val Ser Gly Asp Trp Gly Gly Ile Ile Pro Ala 260 265 270 Met Leu Asn Ser Leu Leu Ala Leu Lys Val Leu Gly Tyr Asp Val Asn 275 280 285 Asp Leu Tyr Val Gln Arg Gly Leu Ala Ala Ile Asp Asn Phe Ala Val 290 295 300 Glu Thr Glu Asp Ser Tyr Ala Ile Gln Ala Cys Val Ser Pro Val Trp 305 310 315 320 Asp Thr Ala Trp Val Val Arg Ala Leu Ala Glu Ala Asp Leu Gly Lys 325 330 335 Asp His Pro Ala Leu Val Lys Ala Gly Gln Trp Leu Leu Asp Lys Gln 340 345 350 Ile Leu Thr Tyr Gly Asp Trp Gln Ile Lys Asn Pro His Gly Glu Pro 355 360 365 Gly Ala Trp Ala Phe Glu Phe Asp Asn Asn Phe Tyr Pro Asp Ile Asp 370 375 380 Asp Thr Cys Val Val Met Met Ala Leu Gln Gly Ile Thr Leu Pro Asp 385 390 395 400 Glu Glu Arg Lys Gln Gly Ala Ile Asn Lys Ala Leu Gln Trp Ile Ala 405 410 415 Thr Met Gln Cys Lys Thr Gly Gly Trp Ala Ala Phe Asp Ile Asp Asn 420 425 430 Asp Gln Asp Trp Leu Asn Gln Leu Pro Tyr Gly Asp Leu Lys Ala Met 435 440 445 Ile Asp Pro Ser Thr Ala Asp Ile Thr Ala Arg Val Val Glu Met Leu 450 455 460 Gly Ala Cys Gly Leu Thr Met Asp Ser Pro Arg Val Glu Arg Gly Leu 465 470 475 480 Thr Tyr Leu Leu Gln Glu Gln Glu Gln Asp Gly Ser Trp Phe Gly Arg 485 490 495 Trp Gly Val Asn Tyr Leu Tyr Gly Thr Ser Gly Ala Leu Ser Ala Leu 500 505 510 Ala Ile Tyr Asp Ala Gln Arg Phe Ala Pro Gln Ile Lys Thr Ala Ile 515 520 525 Ala Trp Leu Leu Ser Cys Gln Asn Ala Asp Gly Gly Trp Gly Glu Thr 530 535 540 Cys Glu Ser Tyr Lys Asn Lys Gln Leu Lys Gly Gln Gly Asn Ser Thr 545 550 555 560 Ala Ser Gln Thr Ala Trp Ala Leu Ile Gly Leu Leu Asp Ala Leu Lys 565 570 575 Tyr Leu Pro Ser Leu Gly Gln Asp Ala Lys Leu Thr Thr Ala Ile Glu 580 585 590 Gly Gly Val Ala Phe Leu Val Gln Gly Gln Thr Pro Lys Gly Thr Trp 595 600 605 Glu Glu Ala Glu Tyr Thr Gly Thr Gly Phe Pro Cys His Phe Tyr Ile 610 615 620 Arg Tyr His Tyr Tyr Arg Gln Tyr Phe Pro Leu Ile Ala Leu Ala Arg 625 630 635 640 Tyr Ser His Leu Gln Ala Ser 645 89 680 PRT Streptomyces coelicolor 89 Met Thr Ala Thr Thr Asp Gly Ser Thr Gly Ala Ser Leu Arg Pro Leu 1 5 10 15 Ala Ala Ser Ala Ser Asp Thr Asp Ile Thr Ile Pro Ala Ala Ala Ala 20 25 30 Gly Val Pro Glu Ala Ala Ala Arg Ala Thr Arg Arg Ala Thr Asp Phe 35 40 45 Leu Leu Ala Lys Gln Asp Ala Glu Gly Trp Trp Lys Gly Asp Leu Glu 50 55 60 Thr Asn Val Thr Met Asp Ala Glu Asp Leu Leu Leu Arg Gln Phe Leu 65 70 75 80 Gly Ile Gln Asp Glu Glu Thr Thr Arg Ala Ala Ala Leu Phe Ile Arg 85 90 95 Gly Glu Gln Arg Glu Asp Gly Thr Trp Ala Thr Phe Tyr Gly Gly Pro 100 105 110 Gly Glu Leu Ser Thr Thr Ile Glu Ala Tyr Val Ala Leu Arg Leu Ala 115 120 125 Gly Asp Ser Pro Glu Ala Pro His Met Ala Arg Ala Ala Glu Trp Ile 130 135 140 Arg Ser Arg Gly Gly Ile Ala Ser Ala Arg Val Phe Thr Arg Ile Trp 145 150 155 160 Leu Ala Leu Phe Gly Trp Trp Lys Trp Asp Asp Leu Pro Glu Leu Pro 165 170 175 Pro Glu Leu Ile Tyr Phe Pro Thr Trp Val Pro Leu Asn Ile Tyr Asp 180 185 190 Phe Gly Cys Trp Ala Arg Gln Thr Ile Val Pro Leu Thr Ile Val Ser 195 200 205 Ala Lys Arg Pro Val Arg Pro Ala Pro Phe Pro Leu Asp Glu Leu His 210 215 220 Thr Asp Pro Ala Arg Pro Asn Pro Pro Arg Pro Leu Ala Pro Val Ala 225 230 235 240 Ser Trp Asp Gly Ala Phe Gln Arg Ile Asp Lys Ala Leu His Ala Tyr 245 250 255 Arg Lys Val Ala Pro Arg Arg Leu Arg Arg Ala Ala Met Asn Ser Ala 260 265 270 Ala Arg Trp Ile Ile Glu Arg Gln Glu Asn Asp Gly Cys Trp Gly Gly 275 280 285 Ile Gln Pro Pro Ala Val Tyr Ser Val Ile Ala Leu Tyr Leu Leu Gly 290 295 300 Tyr Asp Leu Glu His Pro Val Met Arg Ala Gly Leu Glu Ser Leu Asp 305 310 315 320 Arg Phe Ala Val Trp Arg Glu Asp Gly Ala Arg Met Ile Glu Ala Cys 325 330 335 Gln Ser Pro Val Trp Asp Thr Cys Leu Ala Thr Ile Ala Leu Ala Asp 340 345 350 Ala Gly Val Pro Glu Asp His Pro Gln Leu Val Lys Ala Ser Asp Trp 355 360 365 Met Leu Gly Glu Gln Ile Val Arg Pro Gly Asp Trp Ser Val Lys Arg 370 375 380 Pro Gly Leu Pro Pro Gly Gly Trp Ala Phe Glu Phe His Asn Asp Asn 385 390 395 400 Tyr Pro Asp Ile Asp Asp Thr Ala Glu Val Val Leu Ala Leu Arg Arg 405 410 415 Val Arg His His Asp Pro Glu Arg Val Glu Lys Ala Ile Gly Arg Gly 420 425 430 Val Arg Trp Asn Leu Gly Met Gln Ser Lys Asn Gly Ala Trp Gly Ala 435 440 445 Phe Asp Val Asp Asn Thr Ser Ala Phe Pro Asn Arg Leu Pro Phe Cys 450 455 460 Asp Phe Gly Glu Val Ile Asp Pro Pro Ser Ala Asp Val Thr Ala His 465 470 475 480 Val Val Glu Met Leu Ala Val Glu Gly Leu Ala His Asp Pro Arg Thr 485 490 495 Arg Arg Gly Ile Gln Trp Leu Leu Asp Ala Gln Glu Thr Asp Gly Ser 500 505 510 Trp Phe Gly Arg Trp Gly Val Asn Tyr Val Tyr Gly Thr Gly Ser Val 515 520 525 Ile Pro Ala Leu Thr Ala Ala Gly Leu Pro Thr Ser His Pro Ala Ile 530 535 540 Arg Arg Ala Val Arg Trp Leu Glu Ser Val Gln Asn Glu Asp Gly Gly 545 550 555 560 Trp Gly Glu Asp Leu Arg Ser Tyr Arg Tyr Val Arg Glu Trp Ser Gly 565 570 575 Arg Gly Ala Ser Thr Ala Ser Gln Thr Gly Trp Ala Leu Met Ala Leu 580 585 590 Leu Ala Ala Gly Glu Arg Asp Ser Lys Ala Val Glu Arg Gly Val Ala 595 600 605 Trp Leu Ala Ala Thr Gln Arg Glu Asp Gly Ser Trp Asp Glu Pro Tyr 610 615 620 Phe Thr Gly Thr Gly Phe Pro Trp Asp Phe Ser Ile Asn Tyr Asn Leu 625 630 635 640 Tyr Arg Gln Val Phe Pro Leu Thr Ala Leu Gly Arg Tyr Val His Gly 645 650 655 Glu Pro Phe Ala Lys Lys Pro Arg Ala Ala Asp Ala Pro Ala Glu Ala 660 665 670 Ala Pro Ala Glu Val Lys Gly Ser 675 680 90 741 PRT Artificial Sequence Description of Artificial Sequence Figure 6 majority sequence 90 Met Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Gln Leu 1 5 10 15 Val Glu Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Ala Val Ala Arg Ala Leu Asp Arg 50 55 60 Ala Val Asp Tyr Leu Leu Ser Arg Gln Lys Ala Asp Gly Tyr Trp Trp 65 70 75 80 Gly Pro Leu Leu Ser Asn Val Thr Met Glu Ala Glu Tyr Val Leu Leu 85 90 95 Cys His Ile Leu Gly Arg Val Asp Arg Glu Arg Xaa Xaa Met Glu Lys 100 105 110 Ile Arg Arg Tyr Leu Leu His Glu Gln Arg Glu Asp Gly Thr Trp Ala 115 120 125 Leu Tyr Pro Gly Gly Pro Xaa Gly Asp Leu Ser Thr Thr Val Glu Ala 130 135 140 Tyr Val Ala Leu Lys Tyr Leu Gly Xaa Val Ser Ala Asp Glu Pro His 145 150 155 160 Met Val Lys Ala Leu Glu Phe Ile Gln Ser Gln Gly Gly Ile Glu Ser 165 170 175 Ser Arg Val Phe Thr Arg Met Trp Leu Ala Leu Val Gly Glu Tyr Pro 180 185 190 Trp Asp Lys Leu Pro Met Ile Pro Pro Glu Ile Met Leu Leu Pro Lys 195 200 205 Asn Val Pro Leu Asn Ile Tyr Glu Phe Gly Ser Trp Ala Arg Ala Thr 210 215 220 Val Val Pro Leu Ser Ile Val Met Ala Gln Gln Pro Val Xaa Xaa Xaa 225 230 235 240 Xaa Phe Pro Leu Pro Glu Leu Ala Arg Val Pro Glu Leu Tyr Glu Thr 245 250 255 Asp Val Pro Pro Arg Arg Xaa Arg Gly Ala Lys Gly Gly Gly Gly Trp 260 265 270 Xaa Xaa Xaa Ile Phe Asp Ala Xaa Xaa Leu Asp Ser Ala Leu His Gly 275 280 285 Tyr Gln Lys Ala Xaa Xaa Ala Val His Pro Phe Arg Arg Ala Gly Glu 290 295 300 Ala Arg Ala Leu Thr Trp Ile Leu Glu Arg Gln Glu Gly Asp Gly Ser 305 310 315 320 Trp Gly Gly Ile Gln Pro Pro Trp Phe Tyr Ala Leu Ile Ala Leu Lys 325 330 335 Val Leu Gly Met Thr Xaa Gln His Pro Ala Phe Ile Lys Gly Leu Glu 340 345 350 Gly Leu Glu Leu Tyr Gly Val Glu Leu Ser Asp Gly Gly Trp Met Phe 355 360 365 Gln Ala Xaa Ser Ile Ser Pro Val Trp Asp Thr Gly Leu Ala Val Leu 370 375 380 Ala Leu Arg Ala Ala Gly Leu Pro Ala Asp His Pro Ala Leu Val Lys 385 390 395 400 Ala Gly Glu Trp Leu Leu Asp Arg Gln Ile Thr Val Pro Gly Asp Trp 405 410 415 Ala Val Lys Arg Xaa Xaa Pro Asn Leu Lys Pro Gly Gly Trp Ala Phe 420 425 430 Glu Phe Asp Asn Val Asn Tyr Pro Asp Val Asp Asp Thr Ala Val Val 435 440 445 Val Xaa Xaa Xaa Leu Ala Leu Asn Gly Leu Arg Leu Pro Asp Glu Glu 450 455 460 Arg Arg Arg Asp Ala Ile Thr Lys Gly Phe Arg Trp Leu Leu Gly Met 465 470 475 480 Gln Ser Ser Asn Gly Gly Trp Gly Ala Tyr Asp Val Asp Asn Thr Ser 485 490 495 Asp Leu Pro Asn His Leu Pro Xaa Phe Cys Asp Phe Gly Glu Val Xaa 500 505 510 Ile Asp Pro Pro Ser Ala Asp Val Thr Ala His Val Leu Glu Cys Leu 515 520 525 Gly Ser Xaa Xaa Xaa Phe Gly Xaa Xaa Xaa Xaa Xaa Tyr Asp Glu Ala 530 535 540 Trp Lys Val Ile Arg Arg Ala Val Glu Tyr Leu Lys Arg Glu Gln Glu 545 550 555 560 Gln Asp Gly Ser Trp Phe Gly Arg Trp Gly Val Asn Tyr Leu Tyr Gly 565 570 575 Thr Gly Ala Val Val Ser Ala Leu Lys Ala Val Gly Leu Asp Thr Arg 580 585 590 Glu Pro Tyr Ile Gln Lys Ala Leu Asp Trp Leu Glu Ser His Gln Asn 595 600 605 Ala Asp Gly Gly Trp Gly Glu Asp Cys Arg Ser Tyr Glu Xaa Asp Pro 610 615 620 Glu Tyr Ala Gly Gln Gly Ala Ser Thr Ala Ser Gln Thr Ala Trp Ala 625 630 635 640 Leu Met Ala Leu Ile Ala Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Arg 645 650 655 Ala Glu Xaa Xaa Ser Glu Ala Ala Glu Arg Gly Val Ala Tyr Leu Val 660 665 670 Glu Thr Gln Arg Pro Asp Gly Gly Trp Asp Glu Pro Tyr Tyr Thr Gly 675 680 685 Thr Gly Phe Pro Gly Asp Phe Tyr Leu Gly Tyr Thr Met Tyr Arg Gln 690 695 700 Val Phe Pro Leu Leu Ala Leu Gly Arg Tyr Lys Gln Ala Xaa Xaa Xaa 705 710 715 720 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 725 730 735 Glu Arg Xaa Gly Ser 740 91 376 DNA Zygosaccharomyces bailii 91 tgcatggccg ttcttagttg gtggagtgat ttgtctgctt aattgcgata acgaacgaga 60 ccttaaccta ctaaatagta ggtgctagca tttgctggtt tttccacttc ttagagggac 120 tatcggtttc aagccgatgg aagtttgagg caataacagg tctgtgatgc ccttagacgt 180 tctgggccgc acgcgcgcta cactgacgga gccagcgagt ctaaccttgg ccgagaggtc 240 tgggtaatct tgtgaaactc cgtcgtgctg gggatagagc attgtaatta ttgctcttca 300 acgaggaatt cctagtaagc gcaagtcatc aacttgcgtt gattacgtcc ctgccctttg 360 tacacacaag ccgaat 376 92 404 DNA Saccharomyces humaticus 92 ctctttcttg attttgtggg tggtggtgca tggccgttct tagttggtgg agtgatttgt 60 ctgcttaatt gcgataacga acgagacctt aacctactaa atagtggtgc tagcatttgc 120 tggttatcca cttcttagag ggactatcgg tttcaagccg atggaagttt gaggcaataa 180 caggtctgtg atgcccttag acgttctggg ccgcacgcgc gctacactga cggagccagc 240 gagtctaacc ttggccgaga ggtcttggta atcttgtgaa actccgtcgt gctggggata 300 gagcattgta attattgctc ttcaacgagg aattcctagt aagcgcaagt catcagcttg 360 cgttgattac gtccctgccc tttgtacaca ccgcccgtcg ctag 404 93 408 DNA Candida colliculosa 93 ctctttcttg attttgtggg tggtggtgca tggccgttct tagttggtgg agtgatttgt 60 ctgcttaatt gcgataacga acgagacctt aacctactaa atagtggtgc tagcatttgc 120 tggttatcca cttcttagag ggactatcgg tttcaagccg atggaagttt gaggcaataa 180 caggtctgtg atgcccttag acgttctggg ccgcacgcgc gctacactga cggagccagc 240 gagtctaacc ttggccgaga ggtctgggta atcttgtgaa actccgtcgt gctggggata 300 gagcattgta attattgctc ttcaacgagg aattcctagt aagcgcaagt catcagcttg 360 cgttgattac gtccctgccc tttgtacaca ccgcccgtcg ctagtacc 408 94 303 DNA Vitis vinifera 94 ctctttcttg attctatggg tggtggtgca tggccgttct tagttggtgg agcgatttgt 60 ctggttaatt ccgttaacga acgagacctc agcctgctaa ctagctatgt gaaggtgagc 120 ctccgcagcc agcttcttag agggactatg gccgcttagg ccaaggaagt ttgaggcaat 180 aacaggtctg tgatgccctt agatgttctg ggccgcacgc gcgctacact gatgtattca 240 acgagtctat agccttggcc gacaggcccg ggtaatcttt gaaatttcat cgtgatgggg 300 ata 303 95 407 DNA Zygosaccharomyces rouxii 95 ctctttcttg attttgtggg tggtggtgca tggccgttct tagttggtgg agtgatttgt 60 ctgcttaatt gcgataacga acgagacctt aacctactaa atagtggtgc tagcatttgc 120 tggtttttcc acttcttaga gggactatcg gtttcaagcc gatggaagtt tgaggcaata 180 acaggtctgt gatgccctta gacgttctgg gccgcacgcg cgctacactg acggagccaa 240 cgagtctaac cttggccgag aggtctgggt aatcttgtga aactccgtcg tgctggggat 300 agagcattgt aattattgct cttcaacgag gaattcctag taagcgcaag tcatcagctt 360 gcgttgatta cgtccctgcc ctttgtacac accgcccgtc gctagta 407 96 393 DNA Penicillium digitatum 96 gtgctggaat tcggctttgc atggccgttc ttagttggtg gagtgatttg tctgcttaat 60 tgcgataacg aacgagacct cggcccttaa atagcccggt ccgcatttgc gggccgctgg 120 cttcttaagg ggactatcgg ctcaagccga tggaagtgcg cggcaataac aggtctgtga 180 tgcccttaga tgttctgggc cgcacgcgcg ctacactgac agggccagcg agtacatcac 240 cttaaccgag aggtttgggt aatcttgtta aaccctgtcg tgctggggat agagcattgc 300 aattattgct cttcaacgag gaatgcctag taggcacgag tcatcagctc gtgccgatta 360 cgtccctgcc ctttgtacac acaagccgaa ttc 393 97 400 DNA Byssochlamys fulva 97 tgctggaatt cggctttgca tggccgttct tagttggtgg agtgatttgt ctgcttaatt 60 gcgataacga acgagacctc ggctcttaaa tagcccggtc cgcgtttgcg ggccgctggc 120 ttcttagggg gactatcggc tcaagccgat ggaagtgcgc ggcaataaca ggtctgtaat 180 gcccttagat gttctgggcc gcacgcgcgc tacactgaca gggccagcgg gtacatcacc 240 ttggccgaga ggtctgggta atcttgttaa accctgtcgt gctggggata gagcattgca 300 attattgctc ttcaacgagg aatgcctagt aggcacgagt catcagctcg tgccgattac 360 gtccctgccc tttgtacaca caagccgaat tctgcagata 400 98 416 DNA Penicillium chrysogenum 98 tctttcttga tcttttggat ggtggtgcat ggccgttctt agttggtgga gtgatttgtc 60 tgcttaattg cgataacgaa cgagacctcg gcccttaaat agcccggtcc gcatttgcgg 120 gccgctggct tcttaggggg actatcggct caagccgatg gaagtgcgcg gcaataacag 180 gtctgtgatg cccttagatg ttctgggccg cacgcgcgct acactgacag ggccagcgag 240 tacatcacct taaccgagag gtttgggtaa tcttgttaaa ccctgtcgtg ctggggatag 300 agcattgcaa ttattgctct tcaacgagga atgcctagta ggcacgagtc atcagctcgt 360 gccgattacg tccctgccct ttgtacacac cgcccgtcgc tactaccgat tgaatg 416 99 406 DNA Aspergillus nidulans 99 agctctttct tgatcttttg gatggtggtg catggccgtt cttagttggt ggagtgattt 60 gtctgcttaa ttgcgataac gaacgagacc tcggccctta aatagcccgg tccgcgtccg 120 cgggccgctg gcttcttagg gggactatcg gctcaagccg atggaagtgc gcggcaataa 180 caggtctgtg atgcccttag atgttctggg ccgcacgcgc gctacactga cagggccagc 240 gagtacatca ccttggccga gaggcccggg taatcttgtt aaaccctgtc gtgctgggga 300 tagagcattg caattattgc tcttcaacga ggaatgccta gtaggcacga gtcatcagct 360 cgtgccgatt acgtccctgc cctttgtaca caccgcccgt cgctac 406 100 427 DNA Eurotium amstelodami 100 tttcttgatc ttttggatgg tggtgcatgg ccgttcttag ttggtggagt gatttgtctg 60 cttaattgcg ataacgaacg agacctcggc ccttaaatag cccggtccgc atttgcgggc 120 cgctggcttc ttagggggac tatcggctca agccgatgga agtgcgcggc aataacaggt 180 ctgtgatgcc cttagatgtt ctgggccgca cgcgcgctac actgacaggg ccagcgagta 240 catcacctta accgagaggt ctgggtaatc ttgttaaacc ctgtcgtgct ggggatagag 300 cattgcaatt attgctcttc aacgaggaat gcctagtagg cacgagtcat cagctcgtgc 360 cgattacgtc cctgcccttt gtacacaccg cccgtcgcta ctaccgattg aatggctcgg 420 tgaggcc 427 101 442 DNA Aspergillus candidus 101 ctctttcttg atcttttgga tggtggtgca tggccgttct tagttggtgg agtgatttgt 60 ctgcttaatt gcgataacga acgagacctc ggcccttaaa tagcccggtc cgcatttgcg 120 ggccgctggc ttcttagggg gactatcggc tcaagccgat ggaagtgcgc ggcaataaca 180 ggtctgtgat gcccttagat gttctgggcc gcacgcgcgc tacactgaca gggccagcga 240 gtacatcacc ttggccgaga ggtctgggta atcttgttaa accctgtcgt gctggggata 300 gagcattgca attattgctc ttcaacgagg aatgcctagt aggcacgagt catcagctcg 360 tgccgattac gtccctgccc tttgtacaca ccgcccgtcg ctactaccga ttgaatggct 420 cggtgaggcc tccggactgg ct 442 102 407 DNA Gallus gallus 102 ctctttctcg attccgtggg tggtggtgca tggccgttct tagttggtgg agcgatttgt 60 ctggttaatt ccgataacga acgagactct ggcatgctaa ctagttacgc gacccccgag 120 cggtcggcgt ccaacttctt agagggacaa gtggcgttca gccacccgag attgagcaat 180 aacaggtctg tgatgccctt agatgtccgg ggctgcacgc gcgctacact gactggctca 240 gcttgtgtct accctacgcc ggcaggcgcg ggtaacccgt tgaaccccat tcgtgatggg 300 gatcggggat tgcaattatt ccccatgaac gaggaattcc cagtaagtgc gggtcataag 360 ctcgcgttga ttaagtccct gccctttgta cacaccgccc gtcgcta 407 103 407 DNA Triticum aestivum 103 ctctttcttg attctatggg tggtggtgca tggccgttct tagttggtgg agcgatttgt 60 ctggttaatt ccgttaacga acgagacctc agcctgctaa ctagctatgc ggagccatcc 120 ctccgcagct agcttcttag agggactatc gccgtttagg cgacggaagt ttgaggcaat 180 aacaggtctg tgatgccctt agatgttctg ggccgcacgc gcgctacact gatgtattca 240 acgagtatat agccttggcc gacaggcccg ggtaatcttg ggaaatttca tcgtgatggg 300 gatagatcat tgcaattgtt ggtcttcaac gaggaatgcc tagtaagcgc gagtcatcag 360 ctcgcgttga ctacgtccct gccctttgta cacaccgccc gtcgctc 407 104 411 DNA Artificial Sequence Description of Artificial Sequence Figure 7 consensus sequence 104 ctctttcttg attttttggg tggtggtgca tggccgttct tagttggtgg agtgatttgt 60 ctgcttaatt gcgataacga acgagacctc ggcctnctaa atagcncctg tccgnncnna 120 tttgcgggcc ngctggcttc ttagagggac tatcggcntc aagccgatgg aagtttgcgg 180 caataacagg tctgtgatgc ccttagatgt tctgggccgc acgcgcgcta cactgacggg 240 gccagcgagt acataacctt ggccgagagg tctgggtaat cttgtgaaac cctgtcgtgc 300 tggggataga gcattgcaat tattgctctt caacgaggaa tgcctagtag gcgcgagtca 360 tcagctcgtg ttgattacgt ccctgccctt tgtacacacc gcccgtcgct a 411 105 19 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 105 gtggtgctag catttgctg 19 106 17 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 106 ccgctggctt cttaggg 17 107 18 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 107 ggagccagcg agtctaac 18 108 19 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide primer 108 agggccagcg agtacatca 19 109 21 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 109 cggtttcaag ccgatggaag t 21 110 20 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide probe 110 ctcaagccga tggaagtgcg 20 111 1500 DNA Alicyclobacillus acidocaldarius 111 agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60 gggtctcttc ggaggccagc ggcggacggg tgaggaacac gtgggtaatc tgcctttcag 120 gccggaataa cgcccggaaa cgggcgctaa agccggatac gcccgcgagg aggcatcttc 180 ttgcggggga aggcccaatt gggtcgctga gagaggagcc cgcggcgcat tagctagttg 240 gcggggtaac ggcccaccaa ggcgacgatg cgtagccgac ctgagagggt gaccggccac 300 actgggactg agacacggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa 360 tgggcgcaag cctgacggag caacgccgcg tgagcgaaga aggccttcgg gttgtaaagc 420 tctgttgctc ggggagagcg gcatggggga tggaaagccc cgtgcgagac ggtaccgagt 480 gaggaagccc cggctaacta cgtgccagca gccgcggtaa aacgtagggg gcgagcgttg 540 tccggaatca ctgggcgtaa agggtgcgta ggcggtcgag caagtctgga gtgaaagtcc 600 atggctcaac catgggatgg ctttggaaac tgcttgactt gagtgctgga gaggcaaggg 660 gaattccacg tgtagcggtg aaatgcgtag agatgtggag gaataccagt ggcgaargcg 720 ccttgctgga cagtgactga cgctgaggca cgaaagcgtg gggagcaaac aggattagat 780 accctggtag tccacgccgt aaacgatgag tgctaggtgt tggggggaca caccccagtg 840 ccgaaggaaa mccaataagc actccgcctg gggagtacgg tcgcaagact gaaactcaaa 900 ggaattgacg ggggcccgca caagcagtgg agcatgtggt ttaaatcgaa gcaacgcgaa 960 gaaccttacc agggcttgac atccctctga caccctcaga gatgaggggt cccttcgggg 1020 cagaggagac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttcagt 1080 cccgcaacga gcgcaaccct tgacctgtgt taccagcgcg ttgaggcggg gactcacagg 1140 tgactgccgg cgtaagtcgg aggaaggcgg ggatgacgtc aaatcatcat gcccctgatg 1200 tcctgggcta cacacgtgct acaatgggcg gaacaaaggg aggcgaagcc gcgaggcgga 1260 gcgaaaccca aaaagccgct cgtagttcgg attgcaggct gcaactcgcc tgcatgaagc 1320 cggaattgct agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac 1380 acaccgcccg tcacaccacg agagtcggca acacccgaag tcggtgaggt aacccctgtg 1440 gggagccagc cgccgaaggt ggggtcgatg attggggtga agtcgtaaca aggtagccgt 1500 112 1520 DNA Alicyclobacillus acidocaldarius modified_base (236) a, t, c or g 112 agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60 gggtctcttc ggaggccagc ggcggacggg tgaggaacac gtgggtaatc tgcctttcag 120 gccggaataa cgcccggaaa cgggcgctaa tgccggatac gcccgcgagg aggcatcttc 180 ttgcggggga aggcccaatt gggccgctga gagaggagcc cgcggcgcat tagctngttg 240 gcggggtaac ggcccaccaa ggcgacgatg cgtagccgac ctgagagggt gaccggccac 300 actgggactg agacacggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa 360 tgggcgcaag cctgacggag caacgccgcg tgagcgaaga aggccttcgg gttgtaaagc 420 tctgttgctc ggggagagcg gcatggggga tggaaagccc cntgcgagac ggtaccgagt 480 gaggaagccc cggctaacta cgtgccagca gccgcggtaa aacgtagggg gcgagcgttg 540 tccggaatca ctgggcgtaa agggtgcgta ggcggtcgag caagtctgga gtgaaagtcc 600 atggctcaac catgggatgg ctttggaaac tgcttgactt gagtgctgga gaggcaaggg 660 gaattccacg tgtagcggtg aaatgcgtag agatgtggag gaataccagt ggcgaaggcg 720 ccttgctgga cagtgactga cgctgaggca cgaaagcgtg gggagcaaac aggattagat 780 accctggtag tccacgccgt aaacgatgag tgctaggtgt tggggggaca caccccagtg 840 ccgaaggaaa cccaataagc actccgcctg gggagtacgg tcgcaagact gaaactcaaa 900 ggaattgacg ggggcccgca caagcagtgg agcatgtggt ttaattcgaa gcaacgcgaa 960 gaaccttacc agggcttgac atccctctga caccctcaga gatgaggggt cccttcgggg 1020 cagaggagac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttcagt 1080 cccgcaacga gcgcaaccct tgacctgtgt taccagcgcg ttgaggcggg gactcacagg 1140 tgactgccgg cgtaagtcgg aggaaggcgg ggatgacgtc aaatcatcat gcccctgatg 1200 tcctgggcta cacacgtgct acaatgggcg gaacaaaggg aggcgaagcc gcgaggcgga 1260 gcgaaaccca aaaagccgct cgtagttcgg attgcaggct gcaactcgcc tgcatgaagc 1320 cggaattgct agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac 1380 acaccgcccg tcacaccacg agagtcggca acacccgaag tcggtgaggt aacccctgtg 1440 gggagccagc cgccgaaggt ggggtcgatg attggggtga agtcgtaaca aggtagccgt 1500 accggaaggt gcggttggat 1520 113 1497 DNA Alicyclobacillus cycloheptanicus 113 agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60 ggacccttcg gggtcagcgg cggacgggtg agtaacacgt gggtaatctg cccaactgac 120 cggaataacg cctggaaacg ggtgctaatg ccggataggc agcgagcagg catctgctcg 180 ctgggaaagg tgcaaatgca ccgcagatgg aggagcccgc ggcgcattag ctggttggtg 240 gggtaacggc tcaccaaggc gacgatgcgt agccgacctg agagggtgga cggccacact 300 gggactgaga cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg 360 gcgcaagcct gacggagcaa cgccgcgtga gcgaagaagg ccttcgggtt gtaaagctca 420 gtcactcggg aagagcggca aggggagtgg aaagcccctt gagagacggt accgagagag 480 gaagccccgg ctaactacgt gccagcagcc gcggtaatac gtagggggca agcgttgtcc 540 ggaatcactg ggcgtaaagc gtgcgtaggc ggttgcgtgt gtccggggtg aaagtccagg 600 gctcaaccct gggaatgcct tggaaactgc gtaacttgag tgctggagag gcaaggggaa 660 ttccgcgtgt agcggtggaa tgcgtagata tgcggaggaa taccagtggc gaaggcgcct 720 tgctggacag tgactgacgc tgaggcacga aagcgtgggg agcaaacagg attagatacc 780 ctggtagtcc acgccgtaaa cgatgagtgc taggtgttgg ggggtaccac cctcagtgcc 840 gaaggaaacc caataagcac tccgcctggg gagtacggtc gcaagactga aactcaaagg 900 aattgacggg ggcccgcaca agcagtggag catgtggttt aattcgaagc aacgcgaaga 960 accttaccag ggcttgacat ccccctgaca gccgcagaga tgcggtttcc cttcggggca 1020 ggggagacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1080 cgcaacgagc gcaacccttg aactgtgtta ccagcacgtg aaggtgggga ctcacagttg 1140 actgccggcg taagtcggag gaaggcgggg atgacgtcaa atcatcatgc cctttatgtc 1200 ctgggctaca cacgtgctac aatgggcggt acaacgggaa gcgagaccgc gaggtggagc 1260 aaacccctga aagccgttcg tagttcggat tgcaggctgc aactcgcctg catgaagccg 1320 gaattgctag taatcgcgga tcagcatgcc gcggtgaatc cgttcccggg ccttgtacac 1380 accgcccgtc acaccacgag agtcggcaac acccgaagtc ggtggggtaa cccgtcaggg 1440 ggccagccgc cgaaggtggg gttgatgatt ggggtgaagt cgtaacaagg tagccgt 1497 114 1517 DNA Alicyclobacillus cycloheptanicus 114 agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60 ggacccttcg gggtcagcgg cggacgggtg agtaacacgt gggtaatctg cccaactgac 120 cggaataacg cctggaaacg ggtgctaatg ccggataggc agcgagcagg catctgctcg 180 ctgggaaagg tgcaaatgca ccgcagatgg aggagcccgc ggcgcattag ctggttggtg 240 gggtaacggc tcaccaaggc gacgatgcgt agccgacctg agagggtgga cggccacact 300 gggactgaga cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg 360 gcgcaagcct gacggagcaa cgccgcgtga gcgaagaagg ccttcgggtt gtaaagctca 420 gtcactcggg aagagcggca aggggagtgg aaagcccctt gagagacggt accgagagag 480 gaagccccgg ctaactacgt gccagcagcc gcggtaatac gtagggggca agcgttgtcc 540 ggaatcactg ggcgtaaagc gtgcgtaggc ggttgcgtgt gtccggggtg aaagtccagg 600 gctcaaccct gggaatgcct tggaaactgc gtaacttgag tgctggagag gcaaggggaa 660 ttccgcgtgt agcggtggaa tgcgtagata tgcggaggaa taccagtggc gaaggcgcct 720 tgctggacag tgactgacgc tgaggcacga aagcgtgggg agcaaacagg attagatacc 780 ctggtagtcc acgccgtaaa cgatgagtgc taggtgttgg ggggtaccac cctcagtgcc 840 gaaggaaacc caataagcac tccgcctggg gagtacggtc gcaagactga aactcaaagg 900 aattgacggg ggcccgcaca agcagtggag catgtggttt aattcgaagc aacgcgaaga 960 accttaccag ggcttgacat ccccctgaca gccgcagaga tgcggtttcc cttcggggca 1020 ggggagacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1080 cgcaacgagc gcaacccttg aactgtgtta ccagcacgtg aaggtgggga ctcacagttg 1140 actgccggcg taagtcggag gaaggcgggg atgacgtcaa atcatcatgc cctttatgtc 1200 ctgggctaca cacgtgctac aatgggcggt acaacgggaa gcgagaccgc gaggtggagc 1260 aaacccctga aagccgttcg tagttcggat tgcaggctgc aactcgcctg catgaagccg 1320 gaattgctag taatcgcgga tcagcatgcc gcggtgaatc cgttcccggg ccttgtacac 1380 accgcccgtc acaccacgag agtcggcaac acccgaagtc ggtggggtaa cccgtcaggg 1440 ggccagccgc cgaaggtggg gttgatgatt ggggtgaagt cgtaacaagg tagccgtatc 1500 ggaaggtgcg gttggat 1517 115 770 DNA Alicyclobacillus acidoterrestris modified_base (549)..(549) a, c, g, or t 115 gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc gagcccttcg gggctagcgg 60 cggacgggtg agtaacacgt gggcaatccg cctttcagac tggaataaca ctcggaaacg 120 ggtgctaatg ccggataata cacgggtagg catctacttg tgttgaaaga tgcaactgca 180 tcgctgagag aggagcccgc ggcgcattag ctagttggtg aggtaacggc tcaccaaggc 240 gacgatgcgt agccgacctg agagggtgac cggccacact gggactgaga cacggcccag 300 actcctacgg gaggcagcag tagggaatct tccgcaatgg gcgcaagcct gacggagcaa 360 cgccgcgtga gcgaagaagg ccttcgggtt gtaaagctct gttgctcggg gagagcgaca 420 aggagagtgg aaagctcctt gtgagacggt accgagtgag gaagccccgg ctaactacgt 480 gccagcagcc gcggtaatac gtagggggca agcgttgtcc ggaatcactg gggcgtaaag 540 cgtgcgtang cggttgtgta agtctgaact gaaagtccaa ggctcnacct tgggnatgct 600 ttggaaactg catggacttg agtgctggag aggcnaggcn aattccncgt gttaccggtg 660 naaatgcgnt anatatgtgg aggaatacca gtggcnaang cgcctttgct ggacagtgga 720 ctgacgctga aggcacgaaa ancgtgggga ncaacnggat tanatccccn 770 116 1514 DNA Alicyclobacillus acidoterrestris 116 agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60 gagcccttcg gggctagcgg cggacgggtg agtaacacgt gggcaatctg cctttcagac 120 tggaataaca ctcggaaacg ggtgctaatg ccggataata cacgggtagg catctacttg 180 tgttgaaaga tgcaactgca tcgctgagag aggagcccgc ggcgcattag ctagttggtg 240 aggtaacggc tcaccaaggc gacgatgcgt agccgacctg agagggtgac cggccacact 300 gggactgaga cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg 360 gcgcaagcct gacggagcaa cgccgcgtga gcgaagaagg ccttcgggtt gtaaagctct 420 gttgctcggg gagagcgaca aggagagtgg aaagctcctt gtgagacggt accgagtgag 480 gaagccccgg ctaactacgt gccagcagcc gcggtaatac gtagggggca agcgttgtcc 540 ggaatcactg ggcgtaaagc gtgcgtaggc ggttgtgtaa gtctgaagtg aaagtccaag 600 gctcaacctt gggattgctt tggaaactgc atgacttgag tgctggagag gcaaggggaa 660 ttccacgtgt agcggtgaaa tgcgtagata tgtggaggaa taccagtggc gaaggcgcct 720 tgctggacag tgactgacgc tgaggcacga aagcgtgggg agcaaacagg attagatacc 780 ctggtagtcc acgccgtaaa cgatgagtgc taggtgttgg ggggacacac cccagtgccg 840 aaggaaaccc aataagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga 900 attgacgggg gcccgcacaa gcagtggagc atgtggttta attcgaagca acgcgaagaa 960 ccttaccagg gcttgacatc cctctgaccg gtgcagagat gtaccttccc ttcggggcag 1020 aggagacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1080 gcaacgagcg caacccttga tctgtgttac cagcacgtag aggtggggac tcacaggtga 1140 ctgccggcgt aagtcggagg aaggcgggga tgacgtcaaa tcatcatgcc ctttatgtcc 1200 tgggctacac acgtgctaca atgggcggta caacgggaag cgaagccgcg aggtggagca 1260 aaacctaaaa agccgttcgt agttcggatt gcaggctgca actcgcctgc atgaagccgg 1320 aattgctagt aatcgcggat cagcatgccg cggtgaatcc gttcccgggc cttgtacaca 1380 ccgcccgtca caccacgaga gtcggcaaca cccgaagtcg gtgaggtaac cgttatggag 1440 ccagccgccg aaggtggggt tgatgattgg ggtgaagtcg taacaaggta gccgtatcgg 1500 aaggtgcggt tgga 1514 117 1492 DNA Clostridium elmenteitii 117 agagtttgat cctggctcag gatgaacgct ggcggcgtgc ctaacacatg caagtcgagc 60 ggagtgcctt tttggacatt ttcggatgga agaagaggtt acttagcggc ggacgggtga 120 gtaacgcgtg ggcaaccaac cttgatcagg gggacaacat tgggaaacca gtgctaatac 180 cgcatagctc tatattatgg catcatgaga tagagaaaga tttatcggat caagacgggc 240 ccgcgtctga ttagctagtt ggtaaggtaa cggcttacca aggccttgat cagtagccga 300 cctgagaggg tgaccggcca cactggaact gagacacggt ccagactcct acgggaggca 360 gcagtgggga atattgcaca atgggggaaa ccctgatgca gcaacgccgc gtgagcgaag 420 aaggccttcg ggtcgtaaag ctctgtccta tgggaagaag gagtgacggt accataggag 480 gaagccccgg ctaactacgt gccagcagcc gcggtaatac gtagggggca agcgttatcc 540 ggaatcactg ggcgtaaagg gtgcgtaggc ggctaagtaa gtcaggggtg aaaggctacg 600 gctcaaccgt agtaagcctt tgaaactgct tagcttgagt gcaggagagg taagtggaat 660 tcctagtgta gcggtgaaat gcgtagatat taggaggaac accagtggcg aaggcgactt 720 actggactgt aactgacgct gaggcacgaa agcgtgggag cgaacaggat tagataccct 780 ggtagtccac gccgtaaacg atgagtgcta ggtgttgggg gtcaaacctc agtgccggag 840 caaacgcaat aagcactccg cctggggagt acgctcgcaa gagtgaaact caaaggaatt 900 gacgggggac ccgcacaagc agcggagcat gtggtttaat tcgaagcaac gcgaagaacc 960 ttacctgagc ttgacatccc tctgaccggt gagtaaagtc acctttcctt cgggacagag 1020 gagacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc 1080 aacgagcgca acccctgtca ttagttgcca gcatttcgga tgggcactct aatgagactg 1140 ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgttcag 1200 ggctacacac gtgctacaat ggccgataca aagggcagcg aaggagcaat ccggagcgaa 1260 ccccataaag tcggtcccag ttcggattga gggctgcaac tcgcccccat gaagttggag 1320 ttgctagtaa tcgcgaatca gcatgtcgcg gtgaatgcgt tcccgggtct tgtacacacc 1380 gcccgtcaca ccacggaagt cggaagcacc cgaagcccgt taccgaacct tcgggacgga 1440 acggtcgaag gtgaagccga taactggggt gaagtcgtaa caaggtatcc gt 1492 118 1548 DNA Geobacillus subterraneus 118 gagtttgatc ctggctcagg acgaacgctg gcggcgtgcc taatacatgc aagtcgagcg 60 gaccgaatga gagcttgctc ttatttggtc agcggcggac gggtgagtaa cacgtgggca 120 acctgcccgc aagaccggga taactccggg aaaccggagc taataccgga taacaccgaa 180 gaccgcatgg tcttcggttg aaaggcggcc tttggctgtc acttgcggat gggcccgcgg 240 cgcattagct agttggtgag gtaacggctc accaaggcga cgatgcgtag ccggcctgag 300 agggtgaccg gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagta 360 gggaatcttc cgcaatggac gaaagtctga cggagcgacg ccgcgtgagc gaagaaggcc 420 ttcgggtcgt aaagctctgt tgtgagggac gaaggagcgc cgtttgaaca aggcggcgcg 480 gtgacggtac ctcacgagaa agccccggct aattacgtgc cagcagccgc ggtaatacgt 540 agggggcgag cgttgtccgg aattattggg cgtaaagcgc gcgcaggcgg ttccttaagt 600 ctgatgtgaa agcccacggc tcaaccgtgg agggtcattg gaaactgggg gacttgagtg 660 caggagagga gagcggaatt ccacgtgtag cggtgaaatg cgtagagatg tggaggaaca 720 ccagtggcga aggcggctct ctggcctgta actgacgctg aggcgcgaaa gcgtggggag 780 caaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta agtgttagag 840 gggtcacacc ctttagtgct gcagctaacg cgataagcac tccgcctggg gagtacggcc 900 gcaaggctga aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt 960 aattcgaagc aacgcgaaga accttaccag gtcttgacat cccctgacaa cccaagagat 1020 tgggcgttcc cccttcgggg ggacagggtg acaggtggtg catggttgtc gtcagctcgt 1080 gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttgcctcta gttgccagca 1140 ttcagttggg cactctagag ggactgccgg cgaaaagtcg gaggaaggtg gggatgacgt 1200 caaatcatca tgccccttat gacctgggct acacacgtgc tacaatgggc ggtacaaagg 1260 gctgcgaacc cgcgaggggg agcgaatccc aaaaagccgc tctcagttcg gattgcaggc 1320 tgcaactcgc ctgcatgaag ccggaatcgc tagtaatcgc ggatcagcat gccgcggtga 1380 atacgttccc gggccttgta cacaccgccc gtcacaccac gagagcttgc aacacccgaa 1440 gtcggtgagg taacccttac gggagccagc cgccgaaggt ggggcaagtg attggggtga 1500 agtcgtaaca aggtagccgt accggaaggt gcggctggat cacctcct 1548 119 1496 DNA Sulfobacillus disulfidooxidans 119 agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60 ggactcctac gggagtgagc ggcggacggg tgaggaacac gtgggcaatc tgcccattgg 120 actggaataa cgcctggaaa cgggtgctaa ggccagatag acacagaaga ggcctctctt 180 gtgtgggaaa gatgctacgg catcgccagt ggaggagccc gcggcgcatt agctggttgg 240 cggggtaacg gaccaccaag gcgacgatgc gtagccgacc tgagagggtg aacggccaca 300 ctgggactga gacacggccc agactcctac gggaggcagc agtagggaat cttccgcaat 360 gggcgcaagc ctgacggagc aacgccgcgt aagcgaagaa ggccttcggg ttgtaaagct 420 tagtcactcg ggaagagcgg gtgggagagg gaatgctccc accgagacgg taccgggaga 480 ggaagccccg gcaaactacg tgccagcagc cgcggtaata cgtagggggc aagcgttgtc 540 cggaatcact gggcgtaaag ggtgcgtagg cggtgttgtg ggtctgaggt gaaaggtcgg 600 ggctcaaccc tgagaatgcc ttggaaactg caagacttga gtgctggaga ggcaagggga 660 attccacgtg tagcggtgaa atgcgtagag atgtggagga ataccagtgg cgaaggcgcc 720 ttgctggaca gtgactgacg ctgaggcacg aaagcgtggg gagcaaacag gattagatac 780 cctggtagtc cacgccgtaa acgatgagtg ctaggtgttg gggggtacca ccctcagtgc 840 cgaaggaaac ccaataagca ctccgcctgg ggagtacggt cgcaagactg aaactcaaag 900 gaattgacgg gggcccgcac aagcagtgga gcatgtggtt taattcgaag caacgcgaag 960 aaccttacca gggcttgaca tcccccagac gggtgtagag atacaccgtc ccttcggggc 1020 tggggagaca ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc 1080 ccgcaacgag cgcaaccctt gatcggtgtt accagcgcgt aaaggcgggg actcaccggt 1140 gactgccgtc gtaagacgga ggaaggcggg gatgacgtca aatcatcatg ccccttatgt 1200 cctgggcgac acacgtgcta caatgggcgg cacaacggga cgcgagagag caatctggag 1260 ccaacccctg aaaaccgctc gtagttcgga ttgcaggctg caactcgcct gcatgaagcc 1320 ggaattgcta gtaatcgcgg atcagcatgc cgcggtgaat ccgttcccgg gccttgtaca 1380 caccgcccgt cacaccacga gagtcgacaa cacccgaagt cggtggggta acccgtaagg 1440 gggccagccg ccgaaggtgg ggccgatgat tggggtgaag tcgtaacaag gtagcc 1496 120 1428 DNA Bacillus thermoleovorans modified_base (901)..(902) a, t, c or g 120 gagagcttga tcctggctca ggacgaacgc tggcggcgtg cctaatacat gcaagtcgga 60 ccaaatcgga gcttgctctg atttggtcag cggcggacgg gtgagtaaca cgtgggcaac 120 ctgcccgcaa gaccgggata actccgggaa accggagcta ataccggata acaccgaaga 180 ccgcatggtc tttggttgaa aggcggcttt ggctgtcact tgcggatggg cccgcggcgc 240 attagctagt tggtgaggta acggctcacc aaggcgacga tgcgtagccg gcctgagagg 300 gtgaccggcc acactgggac tgagacacgg cccagactcc tacgggaggc agcagtaggg 360 aatcttccgc aatgggcgaa agcctgacgg agcgacgccg cgtgagcgaa gaaggccttc 420 gggtcgtaaa gctctgttgt gagggacgaa ggagcgccgt tcgaagaggg cggcgcggtg 480 acggtacctc acgaggaagc cccggctaac tacgtgccag cagccgcggt aatacgtagg 540 gggcgagcgt tgtccggaat tattgggcgt aaagcgcgcg caggcggttc cttaagtctg 600 atgtgaaagc ccacggctca accgtggagg gtcattggaa actgggggac ttgagtgcag 660 gagaggagag cggaattcca cgtgtagcgg tgaaatgcgt agagatgtgg aggaacacca 720 gtggcgaagg cggctctctg gcctgcaact gacgctgagg cgcgaaagct ggggagcaaa 780 caggattaga taccctggta gtccacgccg taaacgatga gtgctaagtg ttagaggggt 840 cacacccttt agtgctgcag taacgcgata agcactccgc ctggggagta cggccgcaag 900 nntgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 960 gaagcaacgc gaagaacctt accaggtctt gacatcccct gacaacccaa gagattgggc 1020 gttccttcgg gggacagggt gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga 1080 tgttgggtta agtcccgcaa cgcgcgcaac cctcgcctct agttgccagc acgaaggtgg 1140 gcactctaga gggactgccg gtgacaagtc ggaggaaggt ggggatgacg tcaaatcatc 1200 atgcccctta tgacctgggc tacacacgtg ctacaatggg cggtacaaag ggctgcgaac 1260 ccgcgagggg gagcgaatcc caaaaagccg ctctcagttc ggattgcagg ctgcaactcg 1320 cctgcatgaa gccggaatcg ctagtaatcg cggatcagca tgccgcggtg aatacgttcc 1380 cgggccttgt acacaccgcc cgtcacacca cgagagctcg caacaccc 1428 121 1528 DNA Artificial Sequence Description of Artificial Sequence Figure 8 consensus sequence 121 agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60 ggaccncttc ggnggtcagc ggcggacggg tgagtaacac gtgggcaatc tgccnnncag 120 accggaataa cnccnggaaa cgggtgctaa tgccggatan nncncgagna ggcatctnct 180 tgnggngaaa ggtgcaantg natcgctgan ngaggagccc gcggcgcatt agctagttgg 240 tgnggtaacg gctcaccaag gcgacgatgc gtagccgacc tgagagggtg accggccaca 300 ctgggactga gacacggccc agactcctac gggaggcagc agtagggaat cttccgcaat 360 gggcgcaagc ctgacggagc aacgccgcgt gagcgaagaa ggccttcggg ttgtaaagct 420 ctgttgctcg ggaagagcgg canggngngt ggaaagcncc ntgngagacg gtaccgagng 480 aggaagcccc ggctaactac gtgccagcag ccgcggtaat acgtaggggg caagcgttgt 540 ccggaatcac tgggcgtaaa gcgtgcgtag gcggttgngt aagtctgnng tgaaagtcca 600 nggctcaacc ntggganngc nttggaaact gcntgacttg agtgctggag aggcaagggg 660 aattccacgt gtagcggtgn aatgcgtaga natgtggagg aataccagtg gcgaaggcgc 720 cttgctggac agtgactgac gctgaggcac gaaagcgtgg ggagcaaaca ggattagata 780 ccctggtagt ccacgccgta aacgatgagt gctaggtgtt ggggggtcac acccncagtg 840 ccgaaggaaa cccaataagc actccgcctg gggagtacgg tcgcaagact gaaactcaaa 900 ggaattgacg ggggcccgca caagcagtgg agcatgtggt ttaattcgaa gcaacgcgaa 960 gaaccttacc agggcttgac atcccnctga canccnnaga gatgcgnnnt cccttcgggg 1020 cagnggagac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt 1080 cccgcaacga gcgcaaccct tganctgtgt taccagcacg tnnaggtggg gactcacagg 1140 tgactgccgg cgtaagtcgg aggaaggcgg ggatgacgtc aaatcatcat gccccttatg 1200 tcctgggcta cacacgtgct acaatgggcg gtacaanggg angcgaancc gcgaggngga 1260 gcnaanccca naaagccgnt cgtagttcgg attgcaggct gcaactcgcc tgcatgaagc 1320 cggaattgct agtaatcgcg gatcagcatg ccgcggtgaa tncgttcccg ggccttgtac 1380 acaccgcccg tcacaccacg agagtcggca acacccgaag tcggtgnggt aacccntnnn 1440 gggngccagc cgccgaaggt ggggnngatg attggggtga agtcgtaaca aggtagccgt 1500 nnnnnnnnnn nnnnnnnnnn nnnnnnnn 1528 122 1576 DNA Artificial Sequence Description of Artificial Sequence Figure 8 consensus sequence 122 nnnnnnnnnn nnnnnnnnnn ngangaacgc tggcggcgtg cctaanacat gcaagtcgnn 60 nnnnnnnnnn nnnngnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnagcgg cggacgggtg 120 agnaacncgt gggnaancnn cnnnnnnnnn nggnanaacn nnnggaaacn ngngctaann 180 ccnnatannn nnnnnnnnnn gcntnnnnnn nnnnngnaag nnnnnnnnng nnnnncnnnn 240 nnngangngc ccgcgncnna ttagctngtt ggnnnggtaa cggnnnacca aggcnnngat 300 nngtagccgn cctgagaggg tgnncggcca cactggnact gagacacggn ccagactcct 360 acgggaggca gcagtnggga atnttncnca atggnngnaa nnctgangna gcnacgccgc 420 gtnagcgaag aaggccttcg ggtngtaaag ctnngtnnnn ngggnnnnag nnnnnnnnnn 480 nnnnnnnnnn nnnnnnnnng acggtaccnn nngagnaagc cccggcnaan tacgtgccag 540 cagccgcggt aanacgtagg gggcnagcgt tntccggaat nantgggncg taaagngngc 600 gnangcggnn nnnnnngtcn gnnntgaaag nnnnnggctc naccntnnnn nnncnttnga 660 aactgnnnnn ncttgagtgc nggagaggnn agnnnaattc cnngtgtnan cggtgnaant 720 gcgnnanana tnnggaggaa naccagtggc naangcgnct nnnctggncn gnnnnnnnnn 780 ctgannnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn gcngnagnnn nnnnnnnnnn 900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnncncg aanancnnnn nnnnnnnnnn 1020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnngggnan nnnnnnnnnn nnnnnnnnnn 1200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1260 nnnnnnnnnn nnnnnnnnnn ncaannggnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1320 nnnnnnnnnn nnnnnnnnnt tnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nntncccnnn nnnnnnnnnn nnnnnnnnnn 1440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1560 nnnnnnnnnn nnnnnn 1576 123 1526 DNA Artificial Sequence Description of Artificial Sequence Figure 8 majority sequence 123 agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60 ggacccggcg gaggtcagcg gcttacgggt gagtaacacg tgggcaatct gcctttcaga 120 ccggaataac gcccggaaac gggtgctaat gccggataac ccgcgaggag gcatcttctt 180 gcggggaaag gtgcaattgc atcgctgagg gaggagcccg cggcgcatta gctagttggt 240 ggggtaacgg ctcaccaagg cgacgatgcg tagccgacct gagagggtga ccggccacac 300 tgggactgag acacggccca gactcctacg ggaggcagca gtagggaatc ttccgcaatg 360 ggcgcaagcc tgacggagca acgccgcgtg agcgaagaag gccttcgggt tgtaaagctc 420 tgttgctcgg gaagagcggc aaggggagtg gaaagcccct tgcgagacgg taccgagtga 480 ggaagccccg gctaactacg tgccagcagc cgcggtaata cgtagggggc aagcgttgtc 540 cggaatcact gggcgtaaag cgtgcgtagg cggttgcgta agtctggggt gaaagtccag 600 ggctcaaccg tgggaatgct ttggaaactg cgtgacttga gtgctggaga ggcaagggga 660 attccacgtg tagcggtgaa atgcgtagat atgtggagga ataccagtgg cgaaggcgcc 720 ttgctggaca gtgactgacg ctgaggcacg aaagcgtggg gagcaaacag gattagatac 780 cctggtagtc cacgccgtaa acgatgagtg ctaggtgttg gggggtcaca ccctcagtgc 840 cgaaggaaac ccaataagca ctccgcctgg ggagtacggt cgcaagactg aaactcaaag 900 gaattgacgg ggcccgcaca agcagtggag catgtggttt aattcgaagc aacgcgaaga 960 accttaccag ggcttgacat ccctctgaca gccgcagaga tgcggtttcc cttcggggca 1020 ggggagacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1080 cgcaacgagc gcaacccttg acctgtgtta ccagcacgtt aaggtgggga ctcacaggtg 1140 actgccggcg taagtcggag gaaggcgggg atgacgtcaa atcatcatgc cccttatgtc 1200 ctgggctaca cacgtgctac aatgggcggt acaacgggaa gcgaagccgc gaggtggagc 1260 gaaccccaaa aagccgctcg tagttcggat tgcaggctgc aactcgcctg catgaagccg 1320 gaattgctag taatcgcgga tcagcatgcc gcggtgaatc cgttcccggg ccttgtacac 1380 accgcccgtc acaccacgag agtcggcaac acccgaagtc ggtgaggtaa cccgtgtagg 1440 gagccagccg ccgaaggtgg ggtcgatgat tggggtgaag tcgtaacaag gtagccgtnn 1500 nnnnnnnnnn nnnnnnnnnn nnnnnn 1526 124 134 DNA Alicyclobacillus acidocaldarius 124 cgtagttcgg attgcaggct gcaactcgcc tgcatgaagc cggaattgct agtaatcgcg 60 gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccacg 120 agagtcggca acac 134 125 134 DNA Alicyclobacillus acidocaldarius 125 cgtagttcgg attgcaggct gcaactcgcc tgcatgaagc cggaattgct agtaatcgcg 60 gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccacg 120 agagtcggca acac 134 126 134 DNA Alicyclobacillus cycloheptanicus 126 cgtagttcgg attgcaggct gcaactcgcc tgcatgaagc cggaattgct agtaatcgcg 60 gatcagcatg ccgcggtgaa tccgttcccg ggccttgtac acaccgcccg tcacaccacg 120 agagtcggca acac 134 127 134 DNA Alicyclobacillus cycloheptanicus 127 cgtagttcgg attgcaggct gcaactcgcc tgcatgaagc cggaattgct agtaatcgcg 60 gatcagcatg ccgcggtgaa tccgttcccg ggccttgtac acaccgcccg tcacaccacg 120 agagtcggca acac 134 128 134 DNA Alicyclobacillus acidoterrestris 128 cgtagttcgg attgcaggct gcaactcgcc tgcatgaagc cggaattgct agtaatcgcg 60 gatcagcatg ccgcggtgaa tccgttcccg ggccttgtac acaccgcccg tcacaccacg 120 agagtcggca acac 134 129 134 DNA Alicyclobacillus acidoterrestris 129 cgtagttcgg attgcaggct gcaactcgcc tgcatgaagc cggaattgct agtaatcgcg 60 gatcagcatg ccgcggtgaa tccgttcccg ggccttgtac acaccgcccg tcacaccacg 120 agagtcggca acac 134 130 134 DNA Clostridium elmenteitii 130 cccagttcgg attgagggct gcaactcgcc cccatgaagt tggagttgct agtaatcgcg 60 aatcagcatg tcgcggtgaa tgcgttcccg ggtcttgtac acaccgcccg tcacaccacg 120 gaagtcggaa gcac 134 131 134 DNA Geobacillus subterraneus 131 ctcagttcgg attgcaggct gcaactcgcc tgcatgaagc cggaatcgct agtaatcgcg 60 gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccacg 120 agagcttgca acac 134 132 134 DNA Sulfobacillus disulfidooxidans 132 ctcagttcgg attgcaggct gcaactcgcc tgcatgaagc cggaatcgct agtaatcgcg 60 gatcagcatg ccgcggtgaa tccgttcccg ggccttgtac acaccgcccg tcacaccacg 120 agagtcgaca acac 134 133 134 DNA Bacillus thermoleovorans 133 ctcagttcgg attgcaggct gcaactcgcc tgcatgaagc cggaatcgct agtaatcgcg 60 gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccacg 120 agagctcgca acac 134 134 1500 DNA Alicyclobacillus acidocaldarius 134 agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60 gggtctcttc ggaggccagc ggcggacggg tgaggaacac gtgggtaatc tgcctttcag 120 gccggaataa cgcccggaaa cgggcgctaa agccggatac gcccgcgagg aggcatcttc 180 ttgcggggga aggcccaatt gggtcgctga gagaggagcc cgcggcgcat tagctagttg 240 gcggggtaac ggcccaccaa ggcgacgatg cgtagccgac ctgagagggt gaccggccac 300 actgggactg agacacggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa 360 tgggcgcaag cctgacggag caacgccgcg tgagcgaaga aggccttcgg gttgtaaagc 420 tctgttgctc ggggagagcg gcatggggga tggaaagccc cgtgcgagac ggtaccgagt 480 gaggaagccc cggctaacta cgtgccagca gccgcggtaa aacgtagggg gcgagcgttg 540 tccggaatca ctgggcgtaa agggtgcgta ggcggtcgag caagtctgga gtgaaagtcc 600 atggctcaac catgggatgg ctttggaaac tgcttgactt gagtgctgga gaggcaaggg 660 gaattccacg tgtagcggtg aaatgcgtag agatgtggag gaataccagt ggcgaargcg 720 ccttgctgga cagtgactga cgctgaggca cgaaagcgtg gggagcaaac aggattagat 780 accctggtag tccacgccgt aaacgatgag tgctaggtgt tggggggaca caccccagtg 840 ccgaaggaaa mccaataagc actccgcctg gggagtacgg tcgcaagact gaaactcaaa 900 ggaattgacg ggggcccgca caagcagtgg agcatgtggt ttaaatcgaa gcaacgcgaa 960 gaaccttacc agggcttgac atccctctga caccctcaga gatgaggggt cccttcgggg 1020 cagaggagac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttcagt 1080 cccgcaacga gcgcaaccct tgacctgtgt taccagcgcg ttgaggcggg gactcacagg 1140 tgactgccgg cgtaagtcgg aggaaggcgg ggatgacgtc aaatcatcat gcccctgatg 1200 tcctgggcta cacacgtgct acaatgggcg gaacaaaggg aggcgaagcc gcgaggcgga 1260 gcgaaaccca aaaagccgct cgtagttcgg attgcaggct gcaactcgcc tgcatgaagc 1320 cggaattgct agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac 1380 acaccgcccg tcacaccacg agagtcggca acacccgaag tcggtgaggt aacccctgtg 1440 gggagccagc cgccgaaggt ggggtcgatg attggggtga agtcgtaaca aggtagccgt 1500 135 1519 DNA Alicyclobacillus acidoterrestris modified_base (549)..(549) a, c, g, or t 135 gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc gagcccttcg gggctagcgg 60 cggacgggtg agtaacacgt gggcaatccg cctttcagac tggaataaca ctcggaaacg 120 ggtgctaatg ccggataata cacgggtagg catctacttg tgttgaaaga tgcaactgca 180 tcgctgagag aggagcccgc ggcgcattag ctagttggtg aggtaacggc tcaccaaggc 240 gacgatgcgt agccgacctg agagggtgac cggccacact gggactgaga cacggcccag 300 actcctacgg gaggcagcag tagggaatct tccgcaatgg gcgcaagcct gacggagcaa 360 cgccgcgtga gcgaagaagg ccttcgggtt gtaaagctct gttgctcggg gagagcgaca 420 aggagagtgg aaagctcctt gtgagacggt accgagtgag gaagccccgg ctaactacgt 480 gccagcagcc gcggtaatac gtagggggca agcgttgtcc ggaatcactg gggcgtaaag 540 cgtgcgtang cggttgtgta agtctgaact gaaagtccaa ggctcnacct tgggnatgct 600 ttggaaactg catggacttg agtgctggag aggcnaggcn aattccncgt gttaccggtg 660 naaatgcgnt anatatgtgg aggaatacca gtggcnaang cgcctttgct ggacagtgga 720 ctgacgctga aggcacgaaa ancgtgggga ncaacnggat tanatccccn aangcgnggg 780 gaagcaaaca ggattagatt cccnttgtag tcccgccccg taancnatga gtacttagtt 840 gttgggggaa cacaccccan tgcggnggaa acccaataag cactccgcct ggggagtgcg 900 gtcncaagac tgaanctcaa aggaattgac gggggcccgc acaagcagtg gagcatntgg 960 tttaattcga agcaacgcga agaaccttac cagggctnga catccctctg accggtgcag 1020 agatgtacct tcccttcggg gcagaggaga caggtggtgc atggttgtcg tcagctcgtg 1080 tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttgatctgtg ttaccagcac 1140 gttgtggtgg ggactcacag gtgactgccg gcgtaagtcg gaggaaggcg gggatgacgt 1200 caaatcatca tgccctttat gtcctgggct acacacgtgc tacaatgggc ggtacaacgg 1260 gaagcgaagc cgcgaggtgg agcaaaacct aaaaagccgt tcgtagttcg gattgcaggc 1320 tgcaactcgc ctgcatgaag ccggaattgc tagtaatcgc ggatcagcat gccgcggtga 1380 atccgttccc gggccttgta cacaccgccc gtcacaccac gagagtcggc aacacccgaa 1440 gtcggtgagg taaccgttat ggagccagcc gccgaaggtg gggttgatga ttggggtgaa 1500 gtcgtaacaa ggtagccgt 1519 136 1497 DNA Alicyclobacillus cycloheptanicus modified_base (967) a, t, c or g 136 agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60 ggacccttcg gggtcagcgg cggacgggtg agtaacacgt gggtaatctg cccaactgac 120 cggaataacg cctggaaacg ggtgctaatg ccggataggc agcgagcagg catctgctcg 180 ctgggaaagg tgcaagtgca ccgcagatgg aggagcccgc ggcgcattag ctggttggtg 240 gggtaacggc tcaccaaggc gacgatgcgt agccgacctg agagggtgga cggccacact 300 gggactgaga cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg 360 gcgcaagcct gacggagcaa cgccgcgtga gcgaagaagg ccttcgggtt gtaaagctca 420 gtcactcggg aagagcggca aggggagtgg aaagcccctt gagagacggt accgagagag 480 gaagccccgg ctaactacgt gccagcagcc gcggtaatac gtagggggca agcgttgtcc 540 ggaatcactg ggcgtaaagc gtgcgtaggc ggttgcgtgt gtccggggtg aaagtccagg 600 gctcaaccct gggaatgcct tggaaactgc gtaacttgag tgctggagag gcaaggggaa 660 ttccgcgtgt agcggtggaa tgcgtagata tgcggaggaa taccagtggc gaaggcgcct 720 tgctggacag tgactgacgc tgaggcacga aagcgtgggg agcaaacagg attagatacc 780 ctggtagtcc acgccgtaaa cgatgagtgc taggtgttgg ggggtaccac cctcagtgcc 840 gaaggaaacc caataagcac tccgcctggg gagtacggtc gcaagactga aactcaaagg 900 aattgacggg ggcccgcaca agcagtggag catgtggttt aattcgaagc aacgcgaaga 960 accttancag ggctcgacat ccccctgaca gccgcagaga tgcggtttcc cttcggggca 1020 ggggagacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1080 cgcaacgagc gcaacccttg aactgtgtta ccagcacgtg aaggtgggga ctcacagttg 1140 actgccggcg taagtcggag gaaggcgggg atgacgtcaa atcatcatgc cctttatgtc 1200 ctgggctaca cacgtgctac aatgggcggt acaacgggaa gcgagaccgc gaggtggagc 1260 aaacccctga aagccgttcg tagttcggat tgcaggctgc aactcgcctg catgaagccg 1320 gaattgctag taatcgcgga tcagcatgcc gcggtgaatc cgttcccggg ccttgtacac 1380 accgcccgtc acaccacgag agtcggcaac acccgaagtc ggtggggtaa cccgtcaggg 1440 agccagccgc cgaaggtggg gttgatgatt ggggtgaagt cgtaacaagg tagccgt 1497 137 1093 DNA Zygosaccharomyces sp. modified_base (14)..(14) a, c, g, or t 137 attgggccct ctanagcatg ctcgacggcc gccagtgtga tggatatctg cagaattcgg 60 ctttgcatgg ccgttcttag ttggtggagt gatttgtctg cttaattgcg ataacgaacg 120 agaccttaac ctactaaata gtggtgctag catttgctgg tttttccacn ttcttagagg 180 gactatcggt ttcaagccga tggaagtttg aggcaataac aggtctgtga tgcccttaga 240 cgttctgggc cgcacgcgcg ctacactgac ggagccagcg agtctaacct tggccgagag 300 gtctgggtaa tcttgtgaaa ctccgtcgtg ctggggatag agcattgtaa ttattgctct 360 tcaacgagga attcctagta agcgcaagtc atcaacttgc gttgattacg tccctgccct 420 ttgtacacac aagccgaatt ccagcacact ggcggccgtt actagtggat ccgagctcgg 480 taccaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 540 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 600 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 660 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 720 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 780 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 840 agaacatgtg agcaaaaggc cagcanangc cagganccgt aaaaggccgc gtgctggcgt 900 tttncntang ctcgccccct gacagcatnc aaaatcgacg ctcagtcnna ngtggcgaac 960 ccgnnggana taagatacnn gcgttncccc tgnanctccn cntggctntc ngntcnancn 1020 gncgntangg aanctgncnc cttcnccttn ggaacnggnn cttnnnnnnn ancngnngnn 1080 nnnnnnnngg nnn 1093 138 1112 DNA Penicillium digitatum modified_base (3)..(3) a, c, g, or t 138 gangncnncc cnnantnnat cctnagcnga gtngnnaagc gcncgttncc ganggagaag 60 nggacaggtn tccgtancgc aggtnnganc aggagagcgc acgagggagc tncaggggga 120 aacgcctggg atcttnatag tccngtcggg ttcnccacnt ctgacttgag cgtcgatttt 180 gtgatgctcg tcagggggcg gagcntatgg aaaacgccag caacgcggcc ttttacggtt 240 cctggcnttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 300 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag 360 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc 420 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc 480 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac 540 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga 600 aacagctatg accatgatta cgccaagctt ggtaccgagc tcggatccac tagtaacggc 660 cgccagtgtg ctggaattcg gctttgcatg gccgttctta gttggtggag tgatttgtct 720 gcttaattgc gataacgaac gagacctcgg cccttaaata gcccggtccg catttgcggg 780 ccgctggctt cttaagggga ctatcggctc aagccgatgg aagtgcgcgg caataacagg 840 tctgtgatgc ccttagatgt tctgggccgc acgcgcgcta cactgacagg gccagcgagt 900 acatcacctt aaccgagagg tttgggtaat cttgttaaac cctgtcgtgc tggggataga 960 gcattgcaat tattgctctt caacgaggaa tgcctagtag gcacgagtca tcagctcgtg 1020 ccgattacgt ccctgccctt tgtacacaca agccgaattc tgcagatatc catcacactg 1080 gcggccgtcg agcatgctnt agagggccca at 1112 139 1094 DNA Byssochlamys fulva modified_base (1)..(131) a, c, g, or t 139 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 120 nnnnnnnnnn ncnnnnggnn nnncncntnn nnnnngnnnn nnnnnnnnnn nntntcnngg 180 gnnngngcnn nngnaaannn ccngcannnn gccnttnnnn gntnnnggcc ntnngnngnc 240 nnnngntcac angttnntcn ngcgntntcc cnngnttnng nggataacng tattnccgcc 300 tnngagtgag ntgataccgc tcgcngcagc cgaacgaccg agcgcagcga gtcagtgagc 360 gaggaagcgg aagagcgcnc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat 420 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 480 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 540 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat 600 tacgccaagc ttggtaccga gctcggatcc actagtaacg gccgccagtg tgctggaatt 660 cggctttgca tggccgttct tagttggtgg agtgatttgt ctgcttaatt gcgataacga 720 acgagacctc ggctcttaaa tagcccggtc cgcgtttgcg ggccgctggc ttcttagggg 780 gactatcggc tcaagccgat ggaagtgcgc ggcaataaca ggtctgtaat gcccttagat 840 gttctgggcc gcacgcgcgc tacactgaca gggccagcgg gtacatcacc ttggccgaga 900 ggtctgggta atcttgttaa accctgtcgt gctggggata gagcattgca attattgctc 960 ttcaacgagg aatgcctagt aggcacgagt catcagctcg tgccgattac gtccctgccc 1020 tttgtacaca caagccgaat tctgcagata tccatcacac tggcggccgt cgagcatgct 1080 ntagagggcc caat 1094 140 878 DNA Artificial Sequence Description of Artificial Sequence Figure 5 majority sequence 140 gggggttgga tgttccaggc ttgtatttct ccagtgtggg atactggctt ggccgtgttg 60 gcgctgcgtt ctgctgggtt tccggccgat catgccgggt tggttaaggc gggtgagtgg 120 ttgttgggtc ggcagattct cgtggctggc gactgggagg ttcgtcgccg gaaggtgaaa 180 ccgggcggtt tggcgtttga gttcgactgc gtgtactacc cggacgtgga cgatacggcg 240 gtggtcgtct tggcgctcaa tggccttcga ttgccggatg aggggcggcg tcgtgacgcc 300 ttgacgcgtg gcttccgttg gtttgtcggg atgcagagtt cgaacggggg ctggggcgca 360 tacgatgtgg acaacacgcg tgatttgccg aatcggattc cgttttgcga cttcggcgaa 420 gtgattgatc cgccgtcgga agacgtcacc gcccacgtgt tggagtgttt cggcagcttt 480 gggtacgacg aggcctggaa ggtgattcgg cgggcggtgg agtatctcaa gggggagcag 540 cggccggatg ggtgctggtt tggtcgctgg ggcgtcaact acgtgtatgg catgggcgcg 600 gtggtttcgg ggctgaaggc ggtcggtgtc gatatgcgtg agccgtgggt tcaaaagtcg 660 ctcgactggg tcgtggagca tcagaatgcg gatggcggct ggggtgaaga ctgccgntcn 720 tacgaggatc cgnnnctcgc gggtcagggc gcgagnacac cgtcgcagac ngcctgggcg 780 ttgatggcgc tcatcgcggg cggcngtgtc gagtcagang ccgcacnncg cggggtccnn 840 tacctnnnng anacgcagcg cgcngatggt ggctgnnn 878 

1. A method for detecting Alicyclobacillus and Geobacillus in a test sample, the method comprising (a) providing an oligonucleotide set comprising: (i) a forward primer of from 15 to 35 nucleotides in length, said forward primer comprising a sequence which is identical to a first consecutive sequence within the sequence of nucleotide position 1327 through nucleotide position 1460 shown in FIG. 1; (ii) a reverse primer of from 15 to 35 nucleotides in length, said reverse primer comprising a sequence which is complementary to the inverse complement of a second consecutive sequence within the sequence of nucleotide position 1327 through nucleotide position 1460 shown in FIG. 1, said second consecutive sequence being downstream from said first consecutive sequence; and (ii) a probe of from at least 15 nucleotides in length, said probe comprising a sequence which is which is identical to a third consecutive sequence within the sequence of nucleotide position 1327 through nucleotide position 1460 shown in FIG. 1, or the complement thereof, and falls between or overlaps with the first and second consecutive sequences; (b) amplifying DNA in the sample with the said primer set and a polymerase chain reaction, and (c) determining the presence of PCR products of step (b), wherein the presence of a PCR product in the sample is indicative of contamination of the test sample by Alicyclobacillus or Geobacillus or both.
 2. The method of claim 1 wherein the forward primer has a sequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO 5, SEQ ID NO 9, SEQ ID NO 13, SEQ ID NO 17, and SEQ ID NO
 20. 3. The method of claim 1 wherein the reverse primer has a sequence selected from the group consisting of SEQ ID NO 4, SEQ ID NO 8, SEQ ID NO 12, SEQ ID NO 16, SEQ ID NO 19, and SEQ ID NO
 23. 4. The method of claim 1 wherein the probe has a sequence selected from the group consisting of SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 18, SEQ ID NO 21, and SEQ ID NO
 22. 5. A method for detecting Alicyclobacillus and Geobacillus in a test sample, the method comprising (a) providing a oligonucleotide set comprising: (i) a forward primer of from 15 to 35 nucleotides in length, said forward primer comprising a sequence which is identical to a first consecutive sequence within the sequence of nucleotide position 334 through nucleotide position 485 shown in FIG. 5; (ii) a reverse primer of from 15 to 35 nucleotides in length, said reverse primer comprising a sequence which is complementary to the inverse complement of a second consecutive sequence within the sequence of nucleotide position 334 through nucleotide position 485 shown in FIG. 5, said second consecutive sequence being downstream from said first consecutive sequence; and (ii) a probe of from at least 15 nucleotides in length, said probe comprising a sequence which is which is identical to a third consecutive sequence within the sequence of nucleotide position 334 through nucleotide position 485 shown in FIG. 5, or the complement thereof, and falls between or overlaps with the first and second consecutive sequences; (b) amplifying DNA in the sample with the said primer set and a polymerase chain reaction, and (c) determining the presence of PCR products of step (b), wherein the presence of a PCR product in the sample is indicative of contamination of the test sample by Alicyclobacillus or Geobacillus or both.
 6. The method of claim 5 wherein the forward primer has a sequence selected from the group consisting of SEQ ID NO 28, and SEQ ID NO
 29. 7. The method of claim 5 wherein the reverse primer has a sequence selected from the group consisting of SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, and SEQ ID NO
 37. 8. The method of claim 5 wherein the probe has a sequence selected from the group consisting of SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, and SEQ ID NO
 33. 9. A method for detecting Alicyclobacillus and Geobacillus in a test sample, the method comprising (a) providing a oligonucleotide set comprising: (i) a forward primer of from 15 to 35 nucleotides in length, said forward primer comprising a sequence which is identical to a first consecutive sequence within the sequence of nucleotide position 752 through nucleotide position 813 shown in FIG. 1; (ii) a reverse primer of from 15 to 35 nucleotides in length, said reverse primer comprising a sequence which is complementary to the inverse complement of a second consecutive sequence within the sequence of nucleotide position 752 through nucleotide position 813 shown in FIG. 1, said second consecutive sequence being downstream from said first consecutive sequence; and (ii) a probe of from at least 15 nucleotides in length, said probe comprising a sequence which is which is identical to a third consecutive sequence within the sequence of nucleotide position 752 through nucleotide position 813 shown in FIG. 1, or the complement thereof, and falls between or overlaps with the first and second consecutive sequences; (b) amplifying DNA in the sample with the said primer set and a polymerase chain reaction, and (c) determining the presence of PCR products of step (b), wherein the presence of a PCR product in the sample is indicative of contamination of the test sample by Alicyclobacillus or Geobacillus or both.
 10. The method of claim 9 wherein the forward primer has a sequence selected from the group consisting of SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, and SEQ ID NO
 43. 11. The method of claim 9 wherein the reverse primer has a sequence selected from the group consisting of SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52, and SEQ ID NO
 53. 12. The method of claim 9 wherein the probe has a sequence selected from the group consisting of SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, and SEQ ID NO
 49. 13. A method for detecting mold or yeast in a test sample, the method comprising (a) providing a oligonucleotide set comprising: (i) a forward primer of from 15 to 35 nucleotides in length, said forward primer comprising a sequence which is identical to a first consecutive sequence within the sequence of nucleotide position 81 through nucleotide position 225 shown in FIG. 8; (ii) a reverse primer of from 15 to 35 nucleotides in length, said reverse primer comprising a sequence which is complementary to the inverse complement of a second consecutive sequence within the sequence of nucleotide position 81 through nucleotide position 225 shown in FIG. 8, said second consecutive sequence being downstream from said first consecutive sequence; and (ii) a probe of from at least 15 nucleotides in length, said probe comprising a sequence which is which is identical to a third consecutive sequence within the sequence of nucleotide position 81 through nucleotide position 225 shown in FIG. 8, or the complement thereof, and falls between or overlaps with the first and second consecutive sequences; (b) amplifying DNA in the sample with the said primer set and a polymerase chain reaction, and (c) determining the presence of PCR products of step (b), wherein the presence of a PCR product in the sample is indicative of contamination of the test sample by mold or yeast or both.
 14. The method of claim 13 wherein the forward primer has a sequence selected from the group consisting of SEQ ID NO 54, and SEQ ID NO
 58. 15. The method of claim 13 wherein the reverse primer has a sequence selected from the group consisting of SEQ ID NO 55, and SEQ ID NO
 59. 16. The method of claim 13 wherein the probe has a sequence selected from the group consisting of SEQ ID NO 56, SEQ ID NO 57, and SEQ ID NO 60
 17. A method for detecting mold or yeast in a test sample, the method comprising (a) providing a oligonucleotide set comprising: (i) a forward primer of from 15 to 35 nucleotides in length, said forward primer comprising a sequence which is identical to a first consecutive sequence within the sequence of nucleotide position 114 through nucleotide position 238 shown in FIG. 8; (ii) a reverse primer of from 15 to 35 nucleotides in length, said reverse primer comprising a sequence which is complementary to the inverse complement of a second consecutive sequence within the sequence of nucleotide position 114 through nucleotide position 238 shown in FIG. 8, said second consecutive sequence being downstream from said first consecutive sequence; and (ii) a probe of from at least 15 nucleotides in length, said probe comprising a sequence which is which is identical to a third consecutive sequence within the sequence of nucleotide position 114 through nucleotide position 238 shown in FIG. 8, or the complement thereof, and falls between or overlaps with the first and second consecutive sequences; (b) amplifying DNA in the sample with the said primer set and a polymerase chain reaction, and (c) determining the presence of PCR products of step (b), wherein the presence of a PCR product in the sample is indicative of contamination of the test sample by mold or yeast or both.
 18. The method of claim 17 wherein the forward primer has a sequence of SEQ ID NO
 61. 19. The method of claim 17 wherein the reverse primer has a sequence of SEQ ID NO
 62. 20. The method of claim 17 wherein the probe has a sequence of SEQ ID NO
 63. 21. The method of claim 1 wherein the primers i.) do not contain runs of more than 5 of the same nucleotide base, ii) do not contain internal palindromic sequences, iii) do not hybridize to one another under stringent conditions, and iv) have 40 to 60 percent G+C content, and wherein said PCR amplification provides a PCR product that is from 50 to 613 nucleotides in length
 22. The method of claim 1, wherein the PCR is quantitative PCR.
 23. The method of claim 1, wherein the PCR is real-time PCR.
 24. A method of detecting the presence of acidic bacteria in a test sample using real time monitoring of a polymerase chain reaction amplification of a target nucleic acid sequence found in the acidic bacteria, said method comprising the steps of (a) adding to the test sample an effective amount of a forward nucleic acid primer and reverse nucleic acid primer and a nucleic acid probe, wherein the forward primer is selected from the group consisting of SEQ ID NO 1, SEQ ID NO 5, SEQ ID NO 9, SEQ ID NO 13, SEQ ID NO 17, and SEQ ID NO 20, and wherein the reverse primer is selected from the group consisting of SEQ ID NO 4, SEQ ID NO 8, SEQ ID NO 12, SEQ ID NO 16, SEQ ID NO 19, and SEQ ID NO 23, and wherein the probe is selected from the group consisting of SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 10, SEQ ID NO 1, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 18, SEQ ID NO 21, and SEQ ID NO 22 and wherein the probe hybridizes to an amplified copy of the target nucleic acid sequence, and wherein the probe is labeled with a marker which emits a signal upon the hybridization of the probe to the target nucleic acid sequence; (b) amplifying the target nucleic acid sequence by polymerase chain reaction; (c) detecting the emitted signal of the sample.
 25. A method of detecting the presence of fungi in a test sample using real time monitoring of a polymerase chain reaction amplification of a target nucleic acid sequence found in the acidic bacteria, said method comprising the steps of (a) adding to the test sample an effective amount of a forward nucleic acid primer and reverse nucleic acid primer and a nucleic acid probe, wherein the forward primer is selected from the group consisting of SEQ ID NO 54, and SEQ ID NO 58 and SEQ ID NO 61, and wherein the reverse primer is selected from the group consisting of SEQ ID NO 55, SEQ ID NO 59, and SEQ ID NO 62, and wherein the probe is selected from the group consisting of SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 60, and SEQ ID NO 63 and wherein the probe hybridizes to an amplified copy of the target nucleic acid sequence, and wherein the probe is labeled with a marker which emits a signal upon the hybridization of the probe to the target nucleic acid sequence; (b) amplifying the target nucleic acid sequence by polymerase chain reaction; (c) detecting the emitted signal of the sample.
 26. A kit for detecting Alicyclobacillus and Geobacillus in a test sample, comprising a set of oligonucleotides comprising: (a) a forward primer of from 15 to 35 nucleotides in length, said forward primer comprising a sequence which is identical to a first consecutive sequence within the sequence of nucleotide position 1327 through nucleotide position 1460 shown in FIG. 1; (b) a reverse primer of from 15 to 35 nucleotides in length, said reverse primer comprising a sequence which is complementary to the inverse complement of a second consecutive sequence within the sequence of nucleotide position 1327 through nucleotide position 1460 shown in FIG. 1, said second consecutive sequence being downstream from said first consecutive sequence; and (c) a probe of from at least 15 nucleotides in length, said probe comprising a sequence which is which is identical to a third consecutive sequence within the sequence of nucleotide position 1327 through nucleotide position 1460 shown in FIG. 1, or the complement thereof, and falls between or overlaps with the first and second consecutive sequences.
 27. A kit for detecting Alicyclobacillus and Geobacillus in a test sample, comprising a set of oligonucleotides comprising: (a) a forward primer of from 15 to 35 nucleotides in length, said forward primer comprising a sequence which is identical to a first consecutive sequence within the sequence of nucleotide position 334 through nucleotide position 485 shown in FIG. 5; (b) a reverse primer of from 15 to 35 nucleotides in length, said reverse primer comprising a sequence which is complementary to the inverse complement of a second consecutive sequence within the sequence of nucleotide position 334 through nucleotide position 485 shown in FIG. 5, said second consecutive sequence being downstream from said first consecutive sequence; and (c) a probe of from at least 15 nucleotides in length, said probe comprising a sequence which is which is identical to a third consecutive sequence within the sequence of nucleotide position 334 through nucleotide position 485 shown in FIG. 5, or the complement thereof, and falls between or overlaps with the first and second consecutive sequences.
 27. A kit for detecting Alicyclobacillus and Geobacillus in a test sample, comprising a set of oligonucleotides comprising: (a) a forward primer of from 15 to 35 nucleotides in length, said forward primer comprising a sequence which is identical to a first consecutive sequence within the sequence of nucleotide position 752 through nucleotide position 813 shown in FIG. 5; (b) a reverse primer of from 15 to 35 nucleotides in length, said reverse primer comprising a sequence which is complementary to the inverse complement of a second consecutive sequence within the sequence of nucleotide position 752 through nucleotide position 813 shown in FIG. 5, said second consecutive sequence being downstream from said first consecutive sequence; and (c) a probe of from at least 15 nucleotides in length, said probe comprising a sequence which is which is identical to a third consecutive sequence within the sequence of nucleotide position 752 through nucleotide position 813 shown in FIG. 5, or the complement thereof, and falls between or overlaps with the first and second consecutive sequences.
 28. A kit for detecting yeast or mold in a test sample, comprising a set of oligonucleotides comprising: (a) a forward primer of from 15 to 35 nucleotides in length, said forward primer comprising a sequence which is identical to a first consecutive sequence within the sequence of nucleotide position 81 through nucleotide position 225 shown in FIG. 8; (b) a reverse primer of from 15 to 35 nucleotides in length, said reverse primer comprising a sequence which is complementary to the inverse complement of a second consecutive sequence within the sequence of nucleotide position 81 through nucleotide position 225 shown in FIG. 8, said second consecutive sequence being downstream from said first consecutive sequence; and (c) a probe of from at least 15 nucleotides in length, said probe comprising a sequence which is which is identical to a third consecutive sequence within the sequence of nucleotide position 81 through nucleotide position 225 shown in FIG. 8, or the complement thereof, and falls between or overlaps with the first and second consecutive sequences.
 29. A kit for detecting yeast or mold in a test sample, comprising a set of oligonucleotides comprising: (a) a forward primer of from 15 to 35 nucleotides in length, said forward primer comprising a sequence which is identical to a first consecutive sequence within the sequence of nucleotide position 114 through nucleotide position 238 shown in FIG. 8; (b) a reverse primer of from 15 to 35 nucleotides in length, said reverse primer comprising a sequence which is complementary to the inverse complement of a second consecutive sequence within the sequence of nucleotide position 114 through nucleotide position 238 shown in FIG. 8, said second consecutive sequence being downstream from said first consecutive sequence; and (c) a probe of from at least 15 nucleotides in length, said probe comprising a sequence which is which is identical to a third consecutive sequence within the sequence of nucleotide position 114 through nucleotide position 238 shown in FIG. 8, or the complement thereof, and falls between or overlaps with the first and second consecutive sequences. 